Fungal contamination in public buildings: A guide to recognition and management

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2 Fungal contamination in public buildings: A guide to recognition and management

3 Fungal Contamination in Public Buildings: A Guide to Recognition and Management Federal-Provincial Committee on Environmental and Occupational Health Copies of this report are available from: Environmental Health Directorate Health Canada Tunney's Pasture Ottawa, Ontario K1A 0L2 June 1995

4 FUNGAL CONTAMINATION IN PUBLIC BUILDINGS: A GUIDE TO RECOGNITION & MANAGEMENT CONTENTS Page Foreword... i Federal-Provincial Working Group on Mycological Air Quality in Public Buildings...ii Executive Summary... iii I. Introduction... 1 II. Human Health Effects of Indoor Fungi... 3 III. Guidelines Summary of Currently Available Guidelines for Mycological Indoor Air Quality Interpretation of Results from the 1993 Health Canada Technical Guide by the Working Group... 6 IV. Recommended Procedures for the Investigation and Interpretation of Indoor Fungal Contamination Background General Principles Sampling Methods for Indoor Fungal Amplifiers Protocols for Investigation of Indoor Fungal Amplifiers The Purpose of These Protocols How to Use These Protocols Symbols and Expressions Protocols Abbreviated Stachybotrys Algorithm V. Remediation and Preventive Maintenance of Buildings Remediation Preventive Maintenance Background Building Design Considerations... 31

5 5.2.3 Implementation of a Preventive Maintenance Program Communications VI. Recommendations for Future Actions and Research VII. References Glossary... 38

6 DETAILED TABLE OF CONTENTS Protocols Page #1: Why Investigate? #2: Building History Investigation #3: Visual Inspection #4: Air Sampling #5: Examination for Evidence of Fungi in Settled Materials: Dust, Biofilms, and Standing Water #6: Dust Vacuum Sampling #7: Microscopic Examination of Mould or Dust #8: Culture of Swabs, Scrapings, or Surface Dust; and Use of Contact Culture Plates #9: Extraordinary Physical Search: Examination of Difficult Sites within Buildings #10: Mould (Non-stachybotrys) Source Location and Clean-up #11: Minimal Stachybotrys, Source Not Located, Likely Indoors #12: Low to High Stachybotrys, Source Not Located, Likely Indoors #13: Minimal Stachybotrys, Source Not Located, Outdoors or Indoors #14: Low to High Stachybotrys, Source Not Located, Outdoors or Indoors #15: Ergosterol or Glucan Sampling #16: Mycotoxin Sampling from Air or Dust #17: Humidifier Water Direct Microscopy #18: Humidifier Water Culture #19: Cytotoxicity Testing... 27

7 List of Appendices Appendix A Human Health Effects of Indoor Fungi Appendix B Indoor Air Quality in Office Buildings: A Technical Guide, Health Canada, 1993 (Pages 48 49) Appendix C Standard Operating Procedure: An Example for the Investigation and Remediation of Indoor Air Quality Contamination by Mycological Agents Appendix D Sampling Methodology for Fungal Bioaerosols and Amplifiers in Cases of Suspected Indoor Mould Proliferation... 65

8 Foreword In the field of environmental health, authorized to review mycological air quality in there are times when heightened awareness of public buildings. To provide a broad a particular issue creates public concern. One perspective of the problem, members were example of this is the recent experience of chosen with specialization in respiratory and Prince Edward Island with respect to indoor occupational medicine, medical microbiology, air quality in its schools. A major issue was occupational health and safety, medical fungal contamination in particular, how to mycology, environmental microbiology, and interpret laboratory data in terms of the effects environmental health. of fungal contamination on human health. The Working Group on Mycological The Federal-Provincial Committee on Air Quality in Public Buildings held a series of Environmental and Occupational Health was six meetings over a 10-month period. asked for assistance in resolving this issue. As Each member was assigned a topic and wrote part of its working mandate, this committee a section of the report, which was then examines health hazards associated with discussed by the group to achieve consensus. chemicals, radiation, environmental microbials, Much more remains to be understood about tobacco, and hazardous products. Depending the significance of indoor air fungi and their on the topic, fulfilling the committee's mandate impact on human health, and several may require the establishment of recommendations for further research are subcommittees, such as the one on drinking contained in this report. water, whose focus is to develop and The Working Group has attempted to continuously update the Canadian Drinking base its final report on existing scientific data, Water Guidelines. When an issue requires a while recognizing that these data are limited report with a specific time-line, working and imperfect. The resulting guidelines are groups are established. general, and considerable judgement is At Prince Edward Island's request, and required to determine how they should be with the support of the Federal-Provincial applied in specific situations. The guidelines Committee on Environmental and emphasize the elimination of fungal Occupational Health, a working group was amplification points and the protection of building occupants. i

9 Federal Provincial Working Group on Mycological Air Quality in Public Buildings Mr. Richard Davies (Chair) Dr. Irvin Broder Division of Environmental Health Gage Research Institute PEI Department of Health & Social Services Department of Medicine P.O. Box 2000 University of Toronto Charlottetown, PEI 223 College Street C1A 7N8 Toronto, Ontario M5T 1R4 Dr. Richard Summerbell Mycology Department Dr. Robert Dales Ontario Ministry of Health Department of Medicine Laboratory Services University of Ottawa 81 Resources Road Ottawa, Ontario Etobicoke, Ontario K1H 8L6 M9P 3T1 Dr. John Kirkbride Dr. David Haldane Occupational Health Sciences Department of Microbiology Health Canada Victoria General Hospital Ottawa, Ontario 1278 Tower Road K1A 0L3 Halifax, Nova Scotia B3H 2Y9 Dr. Tiiu Kauri Environmental Health Directorate Dr. Alfred Dufour Health Canada Environmental Monitoring System Laboratory Ottawa, Ontario U.S. Environmental Protection Agency K1A 0L2 Cincinnati, Ohio U.S.A. Mr. William Robertson Environmental Health Directorate Dr. Kenneth Yu Health Canada Medical Microbiology & Infectious Diseases Ottawa, Ontario Department K1A 0L2 Occupational Health and Safety University of Alberta Mrs. Louise Damant (Secretariat) 106 Education Park Environmental Health Directorate Edmonton, Alberta Health Canada T6G 2R5 Ottawa, Ontario K1A 0L2 ii

10 Executive Summary The health implications of the fungal growth, but causality in these studies has not contamination of indoor air have become an been established. The burden of illness in the issue of increasing concern in recent years. At population attributable to fungi in private the request of the Government of Prince homes and public buildings is not yet known. Edward Island, and with the support of the While the magnitude of the population risk is Federal-Provincial Committee on unknown, it would seem prudent, based on Environmental and Occupational Health, a current evidence, to remediate indoor sources working group was established to develop an conducive to fungal growth. interim guide to assist public health, A guide to the interpretation of fungal occupational health, and building maintenance sampling data was described in Indoor Air officials in the interpretation of fungal Quality in Office Buildings: A Technical contamination data from public buildings with Guide, published by Health Canada in respect to health. The Working Group strongly supports the The guide has been designed to assist approach described in that document, which in the recognition and management of fungal has been used on a regular basis to investigate contamination problems in public buildings. It and solve mycological indoor air quality also strives to further the understanding of the problems. Accordingly, questions on the health significance of fungi detected in the interpretation of data have arisen. In an course of building investigations. This guide attempt to answer these questions, the applies to indoor air in all public buildings, Working Group reviewed this approach and in excluding hospitals and industrial settings. this document proposes slightly revised The Working Group has reviewed the courses of action. Their essence remains the health effects associated with fungal same as stated in the original 1993 document. contamination of indoor air, reviewed existing The guiding principle during indoor air quality guidelines, and provided investigative or remedial actions is to ensure guidance on procedures for the investigation that no procedures contribute to further fungal and interpretation of indoor fungal contamination of the public building, thus contamination and for remediation and minimizing the health and safety risks to preventive maintenance of buildings. occupants. The procedures for the Published literature describing the investigation of possible mycological health impacts of exposure to indoor air fungi contamination of indoor air can be grouped was critically reviewed. Epidemiological broadly into the following phases: studies have consistently detected an association between respiratory symptoms and home dampness and mould iii

11 Assessing the magnitude of health problems A determination of the occurrence and exposure of previously cryptic contaminants and redistribution of such contaminants via the severity of health problems may be obtained HVAC system or by other means. All from discussions with health professionals, individuals within the building should be building engineers, managers, employees, protected from exposure. union representatives, and building A key element of the report is a maintenance staff. Health questionnaires are detailed step-wise protocol to assist sometimes used as a tool to assemble more professionals who may be asked to investigate comprehensive information. a building with a potential fungal amplification problem. This protocol covers investigation of building history, visual inspection, sampling Identifying problems in the building environment and culturing of airborne propagules, Fungal proliferation is most often examination and culturing of materials and found in buildings in which there is excess assays for ergosterol and mycotoxin. A recent moisture, often in the presence of water- reference describing pathogenic and toxigenic damaged material. Investigators should look fungi is also provided. In this protocol, special for areas in buildings where moisture and attention has been placed on the toxigenic substrates may encourage fungal growth fungus Stachybotrys chartarum (=atra), which for example, areas containing cellulosic is frequently isolated from water-damaged materials, air filters, heat exchangers, buildings in Canada. The protocol includes a humidifiers, water sumps, perimeter heating logic chart to aid in the investigation of and cooling units, wetted carpet, porous duct buildings where Stachybotrys chartarum has lining materials, etc. An attempt should be been isolated or where health symptoms made to correlate these conditions with high- suggest its presence. symptom areas and to designate possible hot spots of contamination. Identification of indoor fungal amplifiers Thorough visual inspection of a problem building, combined with some surface samples for microscopic analysis of apparent mould colonies and of deposits in HVAC systems, may obviate the need for air sampling. Where such inspections yield negative results, air sampling should be considered. Destructive testing is necessary when certain structures of the building have to be taken apart in an attempt to locate the source of suspected contamination. During this phase, the contamination status of the building is expected to be altered by the actions taken by the investigative team, possibly through Risk communication Lines of communication with building occupants, workplace health and safety officials, building managers and owners, employers, and union representatives should be established as soon as health complaints related to indoor air quality are received. Staged plans for investigation of the source of the problem should be presented and agreed to by all parties involved. If fungal contamination is detected, discussions should occur on the health risks and remedial measures to be carried out. Building occupants should be kept up to date during the investigative, remedial, and follow-up stages. iv

12 Remedial actions Strategies for the remediation of potential health problems associated with contaminated indoor air, including the indoor air quality problems caused by fungi are importance of the proper design, installation, based on the elimination of conditions that operation, and maintenance of HVAC systems promote the amplification of these potentially to minimize accumulation, amplification, and hazardous organisms. Remediation of fungal dissemination of micro-organisms. Prevention hazards may involve cleaning affected areas, of fungal contamination is one of the most decontaminating the HVAC systems, removing desirable strategies for risk management. contaminated materials, repairing or replacing damaged materials or structures, and modifying the environmental conditions in the Although the report is based on the affected area. During this phase, the data available in the literature, the Working contamination status of the building is Group recognizes that these data may be expected to be altered by the actions taken by limited and imperfect. Nevertheless, the report the investigative team, possibly through should have general usefulness if it is applied disturbance of newly exposed heavy with reasonable judgement to specific concentrations of contaminants and situations. The report emphasizes the redistribution of such contaminants via the elimination of fungal amplification sources and HVAC system. the protection of building occupants. The design, construction, and Much more remains to be understood maintenance of public buildings should about the significance of indoor air fungi and minimize conditions that allow the their impact on human health. Several accumulation, amplification, and dissemination recommendations for further research are of micro-organisms in indoor air. Building contained in this report. maintenance personnel and building managers should be aware of the v

13 I. Introduction The health implications of indoor In the course of the development of the fungal contaminants have become an issue of guidelines, the methodology for collection of increasing concern in recent years. The air samples was critically reviewed. The purpose of these guidelines is to assist in the guidelines are linked to the method of data recognition and the management of fungal collection. Methods may be chosen on the contamination in public buildings. This basis of the limitations and usefulness of the includes an understanding of the significance data collected, the cost and availability of of fungal measurements performed in the equipment, and current practice in Canada. course of building investigations. In the The applicability of these guidelines to results absence of national standards, guidelines derived from other sampling methodologies published by other organizations have been has not, however, been investigated. used to interpret fungal studies in indoor air. The guidelines described in this It is recognized that fungi can cause a document apply to all indoor air environments spectrum of illnesses in humans, ranging from in all buildings, with two exceptions: hospitals, 1 rhinitis to invasive diseases. If associated with as the role of environmental fungi in causing fungal contamination of buildings, relatively opportunistic infection in well defined diseases are considered building- immunocompromised patients was not related illnesses (BRI). The role of fungi is investigated; and industrial settings, which are not well defined in sick building syndrome outside the scope of this document. The range (SBS), but it is this syndrome that most of climates in Canada, together with the frequently prompts building investigations. variability of buildings and the purposes to The result of these building investigations is which they are put, makes it impossible to commonly the isolation of environmental fungi address every circumstance. Adherence to the for which the health risks are not well defined guidelines, however, should minimize the in these circumstances. impact of exposure to fungal materials and The present guidelines have been guide building maintenance personnel to developed with reference to previously address structural and potential environmental 2 published recommendations. The Working problems in buildings. The importance of Group consulted many experts active in the avoidance of fungal growth by ongoing performance and interpretation of mycological preventive maintenance of buildings is testing of indoor air environments. The emphasized. Potential sources of fungal literature concerning the health impact of contamination should be eliminated wherever exposure to indoor air fungi was critically possible, even in the absence of air sampling reviewed. The present guidelines address results exceeding these guidelines, and potential health effects, recommended conditions permitting growth should be procedures for investigation and interpretation investigated. of indoor fungal contamination, remediation, and preventive maintenance. 1

14 The complexity of indoor air mycology should be recognized. Although fungi are known to cause disease, their contribution to the presence of SBS is not well defined. Measurements of fungal materials can be difficult. Symptoms associated with indoor air quality concerns are not specific. The clinical significance of many mycotoxins has not been established in this setting. In addition to these issues, other questions remain, including the degree of bioavailability of toxins in fungal materials and the role of toxins, individually and in combination, in causing illness. The advances being made in our understanding of this field require that these guidelines be given regular review. 2

15 II. Human Health Effects of Indoor Fungi Several excellent reviews have alopecia, flu symptoms, diarrhea, fatigue, compiled the diversity of illnesses caused by dermatitis, malaise, cough, rhinitis, epistaxis, 3 7 fungi. A detailed review of the literature on 8,18,19 and fever. ß-1,3-glucan, a constituent of the human health effects of indoor fungi is fungal cell walls, may be related to dry cough provided in Appendix A. Mediators of disease 20 and irritation of the skin, eyes, and throat. include mycotoxins, allergens, biologically Epidemiological studies from several countries active cell wall components, and polyclonal have demonstrated associations between cell activators. Antigenic properties of fungi questionnaire-reported respiratory symptoms have been implicated in asthma, allergic and questionnaire-reported mould growth in bronchopulmonary aspergillosis, extrinsic the home. This finding has been remarkably 8 12 allergic alveolitis, and humidifier fever. consistent across different climates, societies, Relevant to the residential setting, Tarlo et al. housing characteristics, and scientific reported that 14 of 26 allergic subjects investigators. One Canadian study also showed (rhinitis, asthma) tested positive, in skin prick 21,22 a dose response effect. The odds ratio for 12 tests, to fungi in their homes. cough was 1.61 (95% confidence interval [CI] Mycotoxin-producing fungi are not ) for the presence of one-versus-no uncommon in residential buildings. In a study mould sites and 2.26 (95% CI ) for of 52 Canadian homes, Miller et al. found two-versus-no mould sites. Unlike home evidence of mycotoxin production in three studies, results from the few studies of public 13 homes containing Aspergillus fumigatus. buildings have been inconsistent. It would Trichothecene mycotoxins have also been therefore be prudent, based on current isolated from the ventilation systems of three evidence, to remediate indoor sources 14 sick buildings in Montreal. Stachybotrys conducive to fungal growth. atra, a hydrophilic mould that can produce It is well established that fungi cause 11 highly toxic macrocyclic trichothecenes, was several diseases, such as systemic infections thought to be responsible for chronic health and asthma. However, cases of these diseases 8 problems in a family dwelling. Symptoms associated with fungal exposure in public were consistent with a toxin etiology, toxins buildings are rarely, if ever, reported. On the were isolated from the air, and workers other hand, fungi have been raised as one of apparently became ill while removing the possible causes of SBS, which is frequently contaminated materials. Other adverse human reported. Symptoms of SBS typically include: health effects from mycotoxin inhalation, documented in uncontrolled case reports, eye irritation (itching and watering include renal 15 failure, tremorgenic eyes) 16 encephalopathy, and organic dust toxic nasal irritation, nasal congestion 17 syndrome. Living in a damp and mouldy throat irritation home has been associated with non-specific cough, wheeze complaints such as headache, sore throat, 3

16 hoarseness, changed voice influence of personal characteristics, job- skin irritation (stinging sensation, related factors, and psychosocial factors on itching, dry skin) SBS. They found that being female, job headache category, work functions (handling of nausea carbonless paper, photocopying, work at video drowsiness, tiredness display terminals), and psychosocial factors of reduced mental capacity, mental work (dissatisfaction with supervisors or fatigue colleagues and quantity of work inhibiting job changed sensation of odour or taste. satisfaction) were associated with workrelated mucosal irritation and work-related The symptoms of SBS are non-specific general symptoms, but these factors could not and have been associated with many factors, account for the differences between the including temperature and humidity extremes. buildings as to the prevalence of the (These factors and their sources are listed in symptoms. The building factor (i.e., the indoor Table 1 on page 11 of the report Indoor Air climate) was strongly associated with the Quality in Office Buildings: A Technical prevalence of the symptoms. Thus, in a given 2 23 Guide, Health Canada, ) Broder et al. building, more than one factor may be at play, identified a number of factors associated with and practice has shown that it is usually decreased well-being among office workers. In difficult to attribute symptoms to specific order of descending magnitude, these were agents. Even when the air quality in a building stress in the workplace, female gender, may be deemed unsatisfactory by investigators, exposure to volatile organic compounds the causes of symptoms may be difficult to (VOCs), and other contributing factors. establish, and determining them may become Fungal spore counts were not associated with an exercise of exclusion. 24 decreased well-being. Skov et al. investigated the 4

17 III. Guidelines 3.1 Summary of Currently Available Guidelines for Mycological Indoor Air Quality The following chart is a compilation of guidelines that the Working Group consulted. GENERAL SOURCE ISSUE INSPECTION SAMPLING BASIS GUIDELINE REMEDIATION ADDRESSED PROCEDURES INFORMATION ACGIH Guidelines for the Bioaerosols of interest in On-site investigation; could lead Description of air sampling; Medical Preassessment ACGIH Fungi, p. 8 General remedial actions; Assessment of Bioaerosols indoor air, e.g., fungi, to remedial actions without routine sampling of pp. 1 9 No numerical guidelines* biocides characterized and in the Indoor Environment mycotoxins, bacteria, further investigation bioaerosols not recommended discussed separately endotoxins Indoor Air Quality in Office General technical guide Individual checklists for various Sampling considerations and Guidelines based on Numerical guidelines Individual remediation Buildings contaminants strategies; occurrence of micro- presented; see Appendix B strategies for various Health Canada RCS (4 min) organisms of this document contaminants in non-residential buildings Biological Particles in Investigation of four main Recommendations for different Available samplers and No methods to adequately Values are given for Not applicable Indoor Environments categories of bioaerosols indoor environment studies: analytical methods for air, assess the exposure to representative categories Commission of the (mites, dander from pets, homes, non-industrial dust, and surface sampling biological particles; health (CEC, p. 35), but no health European Communities fungi, bacteria) reviewed effects and occurrence guideline can be set based on (CEC) discussed current knowledge Airborne fungal populations Fungal bioaerosols in non- Not applicable Andersen N6 single-stage Occurrence based Proposed limit of 200 Not applicable in non-residential buildings residential buildings sampler (1 min) and malt 3 CFU/m for fungal in the United States extract agar (2%) aerosols** SPECIES SPECIFIC Guidelines on Assessment S. atra contamination event Evaluation of microbial For routine assessment, air Chronic exposure to Any concentration exceeding Extensive remedial procedures and Remediation of contamination based on sampling for S. atra not airborne S. atra poses a outdoor levels should be for safe removal of S. atra Stachybotrys atra environmental assessment recommended risk of debilitating health considered positive with different levels of New York (visual inspection, bulk sampling, air monitoring) effects containment 29 1 Note: Additional standards: ASHRAE Standard Ventilation for Acceptable Indoor Air Quality ; OSHA Indoor Air Quality Proposed Rule,

18 * 25 ACGIH 1989 reported that fewer than 100 ** Based on results from more than 2000 indoor 3 CFU/m (colony-forming units per cubic metre) 3 and outdoor samples, a 200 CFU/m guideline was considered of no concern, but ACGIH for fungal bioaerosols is recommended, because indicated that a general Threshold Limit 75% of indoor samples yielded fungal Value (TLV) for a concentration of culturable 3 concentrations less than 178 CFU/m. A critical or countable bioaerosols is not scientifically analysis of results is required if pathogenic or supportable at this time. The presence of toxigenic fungi are detected. unusual levels of a toxigenic fungus should trigger environmental sampling for specific 25 toxins. 3.2 Interpretation of Results from the 1993 Health Canada Technical 2 Guide by the Working Group large space may be missed if air is sampled only from the middle of the space. The choice of media is crucial. The fungi observed are heavily dependent on the media used. Health complaints by building Fungal counts should not be occupants may have many diverse etiologies, interpreted in isolation. The detection of which may be identified by visual inspection of pathogenic and toxigenic fungi requires further the building or by knowing the maintenance investigation or remediation. Observed counts history. Identifiable promoters of fungal are heavily influenced by many factors and growth require correction, and any visible have a great deal of variability, making it fungi require removal. difficult to set limits for acceptable levels. Ideally, sampling should be done to Some of these factors include the type of identify a situation that is unhealthy for the medium the sampler used, sampling time, building occupants. Although it is clear that activities of the occupants, season, natural pathogenic and toxigenic fungi can cause ventilation versus ventilation provided by a disease, the health risks associated with a heating, ventilation, and air conditioning given measured level are, for the most part, (HVAC) system, and geographic location. unknown. Rather than trying to quantify health Canadian guidelines were published in risks, sampling can be used to indicate the Indoor Air Quality in Office Buildings: A presence of an indoor fungal amplifier. The 2 Technical Guide in As described in that presence of high fungal concentrations, certain document, the guidelines are based on a large species mixes, and particular individual species data set gathered over a period of several provides the evidence necessary for years using a Reuter centrifugal sampler with experienced indoor air investigators to a four-minute sampling time. These guidelines diagnose an indoor fungal amplifier. have been found useful by workers in the field The nature of the technique used in the and are used on a regular basis. Accordingly, search determines which species are found. questions of interpretation and clarification Stachybotrys atra may be growing in wall have arisen. The guideline statements are listed cavities but may not be found in air samples below with the intention of clarifying these taken in adjacent rooms. Contamination of a issues, small localized area in a 6

19 but the essence of the guidelines remains as 4. The normal air mycoflora is stated in the original document (see Appendix qualitatively similar to and B), which the Working Group strongly quantitatively lower than that of supports. outside air. The number of fungal 1. Bird or bat droppings accumulating in isolates in outdoor air is affected by the sampling technique, the season, air intakes, ducts, and/or rooms weather conditions, activities, etc. frequently contain virulent pathogenic Published data on the range of fungi, such as Cryptococcus normal values in different parts of neoformans and, in some geographic Canada are not available, and those areas, Histoplasma, as well as fungi of that are available may be based on lower virulence such as the toxigenic sampling techniques unlikely to be Aspergillus fumigatus. These applied in modern indoor studies. organisms are not all reliably detected 5. 3 If more than 50 CFU/m (in either by sampling air or droppings; indoor or outdoor air) of a single accumulated droppings should species (other than Cladosporium or automatically be regarded as Alternaria spp.) are detected, there hazardous sources of pathogenic fungi. may be reason for concern. Further Appropriate action should be taken for investigation should be considered if a the safe removal of any accumulations repeat sample confirms the finding and of bird or bat droppings. establishes that it is based on an indoor 2. The persistent presence, demonstrated source. (As the sampling error is high on repeated sampling, of toxigenic for low colony counts, repeated fungi (e.g., Stachybotrys atra, sampling can reduce variability of the toxigenic Aspergillus, Penicillium, and results and assist in distinguishing Fusarium spp.) indicates that further situations that warrant action.) investigation and appropriate action 6. 3 Up to 150 CFU/m is acceptable if should be taken. there is a mixture of species reflective 3. The confirmed presence of one or of the outdoor air spores. Higher more fungal species occurring as a counts suggest dirty or inefficient air significant percentage of a sample in filters or other problems. indoor samples and not similarly 7. 3 Up to 500 CFU/m is acceptable in present in concurrent outdoor samples summer if the species present are is evidence of a fungal amplifier. primarily Cladosporium or other tree Appropriate action should be taken. or leaf fungi. Values higher than this may indicate failure of the filters or contamination in the building. 7

20 8. The visible presence of fungi on 9. The sensitivity of air sampling for the mouldy ceiling tiles, humidifiers, detection of fungal amplifiers is diffusers, air supply ducts, or other imperfect, and false negative results surfaces (including microscopically can occur; some species are detected visible fungi in humidifiers) requires particularly poorly. If a fungal investigation and remedial action amplifier is suspected, other means of regardless of the airborne spore load. investigation should be used, as described elsewhere in this document. 8

21 IV. Recommended Procedures for the Investigation and Interpretation of Indoor Fungal Contamination 4.1 Background A safe workplace is mandated by law 1. PHASE I Assessing the magnitude of health problems and taking the building history: An estimate of the prevalence and in Canada under various legislative severity of health problems may be frameworks. These include various provincial obtained from discussions with managers, occupational health and safety acts, the employees, union representatives, joint Worker's Right-to-Know, the Workplace occupational health and safety committees, Hazardous Materials Information System and building maintenance staff. Advice (WHMIS), the Canada Labour Code, and should be sought from knowledgeable Transport Canada's Transportation of health professionals. Health questionnaires Dangerous Goods (TDG) Act and are sometimes used as a tool to assemble Regulations. It is of paramount importance to more comprehensive information. The have an operating procedure that will protect value of such data is reduced by the fact the health and safety of visitors, occupants, that in so-called healthy buildings, a and the general public, as well as the workers significant minority of occupants will performing their duties in the investigation of describe symptoms that they attribute to possible fungal contamination in public the building environment. During this buildings (excluding hospitals and industrial phase, the contamination status of the workplaces). building is not expected to be altered by the actions taken by the investigative team. 4.2 General Principles 2. PHASE II Identifying problems in the building environment: Fungi require water The guiding principle is to ensure that and nutrients for growth and proliferation. work or procedures during the investigative or They are most often found in buildings in remedial phase do not contribute to further which there is excess moisture, often in the contamination of the public building and do presence of water-damaged material. not endager the health and safety of building Humidity may be high. There may be occupants. This is a multi-stage process, and, visible condensation on windows. as the investigation proceeds, employees and Colonization of walls and other exposed occupants must be kept informed on a surfaces may be visible. There may be a continuing basis. (For further details, see distinctive fungal odour. Investigators Appendix C.) should look for areas in buildings where Procedures for the investigation of moisture and substrates may encourage possible mycological contamination in indoor fungal growth for example, areas air can be grouped broadly into the following containing six phases: 9

22 cellulose material (paper, cardboard, be carried out. Individuals involved in the wood, etc.), air filters, heat exchangers investigation and remediation should have (condensation on cooling coils), received appropriate training as required humidifiers, water sumps, perimeter by the Occupational Health and Safety Act heating and cooling units, wetted carpet, appropriate to the particular province and porous lining materials, etc. An attempt WHMIS. Building occupants should be should be made to correlate these kept up to date during the investigative, conditions with high-symptom areas and remedial, and follow-up stages. Detailed to designate possible hot spots of information on effective communication contamination. During this phase, the strategies for dealing with indoor air contamination status of the building is not 2,32,33 quality problems is available. During expected to be altered by the actions taken this phase, the contamination status of the by the investigative team. building is not expected to be altered by 3. PHASE III Sampling (see also the actions taken by the investigative team. Appendix D): 5. PHASE V Destructive testing: a. Transparent tape surface sampling. Destructive testing occurs when certain b. Scrapings of contaminated materials. structures of the building have to be taken c. Routine air sampling. apart in an attempt to locate the source of d. Bulk sampling. suspected contamination. During this During this phase, the contamination phase, the contamination status of the status of the building is not expected to be building is expected to be altered by the altered by the actions taken by the actions taken by the investigative team, investigative team. possibly through exposure of previously 4. PHASE IV Risk communication: Risk cryptic contaminants and redistribution of communication has been defined as the such contaminants via the HVAC system act of conveying or transmitting or by other means. All individuals within information between interested parties the building should be protected from about the levels of health or environmental exposure. risks; the significance or meaning of such 6. PHASE VI Remedial actions: risks; or decisions, actions or policies a. Removal of contaminated material. aimed at managing or controlling such b. Decontamination of the HVAC and 31 risks. Lines of communication between other systems as required. building occupants, workplace health and c. Repair or replacement of damaged safety officials, building managers and materials and/or structures. owners, employers, and union During this phase, the contamination representatives should be established as status of the building is expected to be soon as health complaints related to indoor altered by the actions taken by the air quality are received. Steps for investigative team, possibly through investigation of the source of the problem disturbance of newly exposed heavy should be presented and agreed to by all concentrations of contaminants parties involved. If fungal contamination is detected, discussions should occur on the health hazards and remedial measures to 10

23 and redistribution of such contaminants via possibility of non-fungal factors being the HVAC system. responsible for the problem must also be 4.3 Sampling Methods for Indoor thoroughly revisited. Because fungi may proliferate out of sight in ducts and other Fungal Amplifiers hidden sites, particularly in larger buildings with complex HVAC systems, a negative Air sampling is very useful in the visual inspection alone does not allow one to assessment of indoor fungal amplification conclude that problem fungal amplifiers are problems, but it must be used strategically. absent. Also, a building may contain both When there is no reason to suspect fungal grossly evident and hidden amplifiers. Air problems, such sampling is unnecessary. For sampling may be the most reliable and example, when a non-fungal cause of air accessible way of detecting fungal amplifiers quality problems is evident in routine analysis, that are not seen in initial visual inspection. air sampling for fungi will generally be Detailed suggestions regarding when and when superfluous. When indoor fungal proliferation not to perform air sampling can be found in is considered possible for example, where Section 4.4. Also, for further details on there is a history of high humidity or water sampling methodology, see Appendix D. damage or where basic air quality tests fail to suggest a likely cause of perceived air problems air sampling may be considered. It is no longer used in isolation, however, and it is not regarded as the sole means of Protocols for Investigation of Indoor Fungal Amplifiers The Purpose of These Protocols assessing the presence of fungi. Often, The following protocols are not thorough visual inspection of a problem intended to be read as background building, combined with some surface samples information, but rather are designed to serve for microscopic analysis of apparent mould as recipes to assist the investigator in colonies and of deposits in ducts, may detecting potentially problematic indoor fungal supplement information obtained in air amplifiers. Each protocol is numbered and is sampling or, in some cases, even render air referred to by number in the text. This device, sampling unnecessary. This is particularly the although necessary for brevity and procedural case when flourishing amplifiers of aerially clarity, may make the text difficult to read dispersing moulds are found in visual discursively, and it is recommended that a inspection, leading to the working assumption copy of the relevant section of the Table of that they are the predominant sources of Contents be made and kept to one side to be indoor-derived inoculum in the building. In used as a guide to the numbers. Each protocol such cases, prompt remediation may eliminate has a preamble partially outlining its rationale. the problem and, if symptoms experienced by The intended user of these protocols is affected persons are alleviated, void the need a knowledgeable member of any of the various for further sampling. Where visual inspections professions (occupational health and safety give negative results (including cases where inspector, building inspector, mycologist, some previously visible mould has been health professional, building cleaned up) but an air quality problem still persists, the prospect of doing air samples must be given further consideration. The 11

24 engineer, occupational hygienist, etc.) who obtained that give a fair approximation of the may be called in to investigate a building with amount of S. chartarum present. In some a possible fungal amplification problem. Such cases, air or dust sampling may yield no a building either may be associated with health S. chartarum at all, yet considerable quantities complaints or may be a place where mould of still-toxic conidia may be evidenced on growth has been noted visually or detected direct examination of dust or may be found through some sort of routine sampling within a wall cavity of a room whose procedure. No information on fungal occupants have been distressed. The episodic identification is provided here. An excellent culturability of this species necessitates several review of the pathogenic and toxigenic fungi atypical search strategies, outlined in protocols known to be of concern has been published by #11 #14. Other common toxigenic indoor 34 Gravesen et al. moulds tend to be culturable, although in a few Although it is mentioned elsewhere in cases growth may be contingent on the water this document, it must be stressed again here activity of the media used. Protocols for these that this material is not intended to assist in the moulds are generally less elaborate. search for opportunistic fungal pathogens in hospitals. In that situation, the search may be How to Use These Protocols directed towards numerically rare types of Begin at #1 and follow the suggestions fungal propagules; in general indoor fungal in that section that direct you to other amplification problems, on the other hand, protocols that may apply. For each protocol numerically abundant propagule types are most you are directed to, read the short introductory often the object of concern. Detection paragraph(s) and then each of the numbered strategies in the two cases are necessarily quite subsections below it (e.g., #8a, #8b, #8c, #8d). different. Choose only the ones that apply to your Several of the protocols are directed situation and follow them, ignoring the ones towards the diagnosis of infestations caused by that do not apply. The subsections will tell you Stachybotrys chartarum (=atra). This which other numbered protocols to go to. particular mould is used as a model for A suggested protocol marked with an developing the algorithm for assessing indoor asterisk (*) is usually a valuable technique that mould proliferation. It is singled out not is very labour intensive, knowledge intensive, because of its relatively high toxicity, but or costly and that would ordinarily be done rather for purely technical reasons: unusual only after simpler techniques had proven techniques are often needed to detect it unable to resolve the situation. Techniques accurately. Unlike other toxigenic moulds marked with an asterisk can therefore associated with indoor proliferation, it ordinarily be ignored in early investigations frequently is represented in the environment and in investigations that turn out to be predominantly by conidia and other materials straightforward, lacking unusual complicating that, although actively toxic, do not grow on factors. fungal culture media. The majority of conidia in many amplifiers appear either to be nonviable or to be otherwise inhibited in germination. Only uncommonly are numbers of colonies 12

25 4.4.3 Symbols and Expressions investigation of public buildings should not Symbols and expressions used in the normally proceed in isolation from various protocols are explained below. investigation of other parameters influencing air quality (see protocol #2) Protocols #1: Why Investigate? a) If symptoms are compatible with those outlined in Chapter 2, then do a visual Investigation of a potential fungal contribution inspection (#3) and check the building to indoor air problems may follow either 1) the history (#2). If these do not reveal an appearance of symptoms compatible with obvious source of the problem, then do air exposure to fungal bioaerosols (see Chapter sampling (#4) or thorough dust sampling 2), or 2) the detection of potentially (#6)*. Also, do examination of settled problematical fungal material in a building materials (#5). environment. Apart from the necessarily rapid diagnosis and clean-up of gross contamination, fungal Symbol/expression Explanation * - optional, thorough protocol, labour intensive, and not always done, but an option available for the most thorough examinations. rely on - do protocol if not done before; if done before, refer to those results. remediation protocol - general protocol for mould clean-up (see Section 5.1). benchmark or - established threshold at which contamination exceeds tolerable action level levels and action must be taken. 13

26 b) If there are no symptoms, take no action Building history (relevant to fungal growth): unless a moderate to large quantity of Inquire about sites associated with symptoms; mould is seen (do #2, #3, #7, #8*, #4*, any previous flooding or other water damage; #5*) or high counts are revealed in seasonal humidity; winter condensation; preliminary or routine air samples (do #2, summer condensation on cold plumbing #3, #5) or Stachybotrys is detected in fixtures and pipes; frequency of carpet preliminary or routine air samples, dust cleaning, duct cleaning, HVAC maintenance; samples, sampled visible mould or cultured humidifier, aerosolizer, or mister type and swabs, surface materials, or contact plates maintenance; and long-time colonization of (proceed according to instructions for attic or other areas by birds or bats. Interpret Stachybotrys in protocol #4 for air any water damage seen in visual inspection samples, #6 for dust samples, #7 for (#3) as a historical indicator. macroscopically or microscopically visible Stachybotrys, and #8 for swab/surface a) The building history suggests there is an material cultures and contact plates). area of readily discernible mould growth #2: Building History Investigation (e.g., water-damaged surfaces or stored materials, poorly maintained ducts or HVAC, etc.); perform a visual inspection Building history (general): Indoor air (#3) wherever this has not been done problems may have various etiologies and already. should not be assumed automatically to be of microbial origin. Important factors such as b) The building history suggests there is a non-fungal chemical contamination (e.g., hidden locus of mould growth (e.g., volatile organic compounds of industrial or previously flooded wall cavity or false commercial origin, tobacco smoke, ozone, ceiling; previously flooded carpet backing; nitrogen dioxide, carbon monoxide) are unshielded cold water pipes or leaky pipes outside the scope of these protocols, as are the in false ceilings; previous fire extinguished potential effects of sociological issues, such as by water, possibly affecting wall cavities; labour/management disputes on perceived insulation materials present inside ducts). environmental tolerance. Also beyond the In these and similar cases, where symptoms scope of these protocols, but possibly germane warrant only, do an extraordinary physical in investigated cases, is the evaluation of search (#9). Where a hidden locus of bacterial diseases, allergies, and intoxications mould growth is suggested but the area is (including endotoxin exposure), viral diseases, not associated with symptoms and is arthropod (e.g., insect, mite) allergies, unlikely to be a source of mould inoculum protozoal allergies, and allergies to any to areas associated with symptoms (e.g., is aerosolized animal and plant parts or products. not connected to vents that may distribute A thorough investigation should consider as mould inoculum to rooms where symptoms many of these factors as may be applicable. have been noted), either take no action or perform #9b*. 14

27 c) The building history suggests long-time a) Visual inspection reveals mould or bird or bat colonization of the attic or suspected mould: take scrapings or elsewhere: inspect visually (#3) while transparent tape mount for direct wearing a high-efficiency particulate air microscopic examination (#7) and (HEPA) filter respirator, or call an expert. scrapings, swabs, or contact plates for culture (#8*). If the attic contains d) The building history is not suggestive, or substantial amounts of bird or bat guano, information is lacking or incomplete; rely consult an expert. on #3, #4 (or #6), #5. Procedures #4 and #6 (air and dust sampling) are relatively b) Visual inspection reveals no mould but labour intensive and should be undertaken reveals damp materials or areas of exposed only where complaints, history, or physical soil as indicated above: rely on #2, #4, and conditions are suggestive of a potential #5. If a substantial area of exposed soil is mould problem, but where the source of found, ensure that any air sampling this problem is not immediately evident includes samples taken near this area to upon walk-through (#3) and microscopic probe its significance as a source of mould (#5, #7) visual inspection, or where the propagules. Sample of soil may also be bioaerosol significance of visually evident analysed using the same techniques as for mould growth is queried. dust analysis (#6*). #3: Visual Inspection c) Visual inspection reveals neither mould nor apparent dampness or substantial indoor Inspect for mould growth or water damage on soils: if symptoms are present in the walls, especially in basements, on window building occupants, rely on #2, #4, #5, and frames, and on carpets (check backing in #6*. If no symptoms are present and no water-stained areas if possible), on ceiling other evidence of indoor fungal tiles, as well as on any currently or formerly amplification exists, take no further action. damp material made of fibrous cellulose If no symptoms are present but other (wallpaper, books, papers, shredded evidence of fungal amplification exists, newspaper insulation) and all accessible consider doing an extended visual HVAC components; also check for any inspection (#3); if a hidden building substantial indoor space with exposed soil maintenance problem is suggested, do an such as unfinished basements or crawl spaces, extraordinary physical search (#9). extensive amount of indoor plants, contiguous greenhouses, attics with resident or seasonal #4: Air Sampling birds, bats, or other animals (wear HEPA filter respirator when checking if building history Vacuum/culture devices such as RCS, (#2) indicates long-term bird or bat breeding Andersen, and slit-to-agar samplers are or roosting in attic); and any other likely recommended for air sampling in public mould sources. buildings; note that current Health Canada guidelines are based on four-minute samples with the RCS. If no such device is available, 15

28 settle plates may be used for preliminary study, It is critical that outdoor controls be taken well but they should be interpreted by an expert away from the building being tested or any familiar with their limitations and with similar buildings and upwind of any possible common local indicator species of indoor air outflows. Heavily contaminated buildings mould proliferation. Even as interpreted by will significantly adulterate outdoor controls such an expert, settle plates may reliably and render them useless unless proper indicate only extreme (very mouldy, very procedures are followed. Outdoor sampling clean) environments (heavy or minimal mould should take place at least 6 m, and preferably growth after one-hour exposure of a sufficient 10 m, windward of the building of concern number of plates), whereas low to moderate (and any similar buildings) if possible; indoor mould growth may give settle plate investigators using electrical devices should results that are difficult to interpret. See carry enough all-weather extension cord to Appendix D for further background facilitate this. Where buildings are closely information on vacuum/culture and settle plate crowded, air sampling on the windward side of sampling. the roof may be tried; however, if high levels of moulds ordinarily associated with indoor In any case where the indoor environment is sources are obtained, further investigation suspected to be the source of mould-related should be undertaken to determine the problems, analysis of air samples should prevalence of these species outdoors in the concentrate on counting and identifying 2 general area (1 km ) of the sample. moulds of indoor origin or indoor accumulation, not transitory moulds of In general, well-established, medically outdoor origin. This is accomplished by a meaningful benchmarks for acceptable indoor species-by-species comparison with control mould levels are lacking; in their absence, the samples of outdoor air. Disregard any species best benchmark to use for the detection of found indoors at a level similar to or lower indoor mould amplifiers is that no species than its contemporaneous outdoor level should be at significantly greater levels indoors (exceptions: Stachybotrys, dimorphic systemic than outdoors (test significance with chifungal pathogens, Cryptococcus neoformans) squared test where applicable). A species more unless other evidence indicates the species is prevalent indoors than outdoors is usually nonetheless proliferating indoors. Species need proliferating indoors. Only rarely will outdoor not be named in these comparisons, but spora sediment out and accumulate indoors to isolates must be recognized as conspecific the extent that species of outdoor origin based on mycological analysis of macroscopic appear to be more common indoors than or microscopic characters. outdoors. This may, however, occur near large outdoor composting facilities. Indoor Identification of Aspergillus species, proliferation usually indicates a maintenance Penicillium subgenera, and genera of other problem, which may also be associated with numerically significant fungi is recommended. some degree of health risk. Minor species present in proportions below 1% can be ignored provided any Stachybotrys, Aspergillus, or other pathogenic or toxigenic fungi known to be of concern are detected. 16

29 Similarly, a species found indoors in a standardized to compose the baseline data, but proportion of total spora much greater than its also the environments studied (e.g., type of outdoor proportion is usually proliferating building, geographic area) should be highly indoors and indicates a maintenance problem comparable. Some tentative benchmarks based (e.g., if Cladosporium cladosporioides makes on airborne fungal levels in essentially up 20% of the outdoor spora and 40% of the proliferation-free, normal Canadian office indoor spora, indoor amplification is likely and buildings were originally proposed by Health is probably occurring in an excessively moist 2 Canada and appear in a slightly revised area). As exposure-related health risk is version in the current document (Section 3.2). currently difficult or impossible to quantify for indoor moulds, a better approach is to use Growth media for air sampling fall into two mould sampling to detect building maintenance physiological categories: high water activity problems and to correct them with the (general fungal growth media such as malt assurance that any genuinely associated, extract agar or colony diameter restricting mould-related health problems will be media such as Littman oxgall or rose bengal corrected at the same time. agar) (see Appendix D), and low water activity (medium with high solute concentration, e.g., Benchmarks or action levels based on absolute dichloran 18% glycerol agar, used for rather than relative numbers are a frequently detecting fungi growing on very dry material discussed scientific ideal, but most such such as dust components and not growing well benchmarks heretofore proposed are based on on general fungal media). Since many common unwarranted assumptions or have arbitrary indoor moulds grow on these low water elements. In recent years, most authorities activity media, their use in indoor mould have declined to give uniform and absolute sampling has increased in recent years. The standards for acceptable fungal propagule usage of various media in indoor fungal levels in indoor air. Indoor/outdoor sampling is further discussed in Appendix D. comparisons are generally superior in the Antibacterial antibiotics are usually detection of potentially problematic incorporated into the media in mycological proliferations. In some cases, counts of moulds studies. of indoor origin in buildings with air quality problems have been compared with average a) Air sampling with vacuum/culture device mould levels in complaint-free buildings. Such shows moulds of indoor origin present and comparisons with normal airspora levels are exceeding the benchmark level (e.g., best interpreted in combination with significantly higher than outdoor levels for indoor/outdoor comparison and amplifier same species; in the case of Stachybotrys, detection as outlined above. At best, studies any isolation is above the benchmark and comparing buildings under investigation with should trigger further investigation). Go to results from unknown problem and non- #10, and, if any Stachybotrys is present, go problem buildings must be interpreted with to #4b #4f and follow the most fitting caution. Not only must technology be highly choice. 17

30 b) Minimal Stachybotrys found indoors but #5: Examination for Evidence of Fungi in not found in outdoor controls. (Minimal = Settled Materials: Dust, Biofilms, and a vanishingly small number detected: for Standing Water example, a single isolated colony found under any circumstances, or two widely Air sampling using culturing techniques has dispersed colonies in a large series of traditionally been favoured over direct samples from different rooms. Note that sampling of potential fungal substrates and colony means an actual fungal colony sediments indoors. This type of air sampling as observed on the sampling medium after the strength that it reflects (to a reasonable 3 incubation, not a CFU/m in calculation.) degree of approximation) the number of live Go to #11. fungal propagules a person's respiratory system is exposed to. Its disadvantages include c) Low to high numbers of Stachybotrys its insensitivity to non-viable propagules, its found ( low to high : at least three reliance on potentially fluctuant airborne colonies in a multi-room sample or two propagule populations, its short sampling colonies from a single room; or more times, making phenomena like diurnal cycles colonies found under any circumstances) of propagule release difficult to discern, and its but either not found in outdoor controls or inability to discern the source of indoor found in outdoor controls at statistically propagules. On the other hand, the direct significantly lower levels (chi-squared test). sampling of sediments and solid or liquid Go to #12. fungal substrata may facilitate the detection of non-viable fungal propagules, the confirmation d) Minimal Stachybotrys found indoors (see of sites of fungal growth, the in situ #4b for definition of minimal) and also in quantification of deleterious fungal chemicals outdoor controls at minimal or higher level. or of chemicals indicative of fungal biomass, Go to #13. and the discernment of patterns of fungal propagule deposition over moderately long e) Low to high (see #4c for definition) periods of time. Note that significant biofilms numbers of Stachybotrys found indoors and and potentially contaminated standing water also in outdoor controls at insignificantly ordinarily should not be present in a building different (chi-squared test) or higher levels. and should ordinarily be remediated Go to #14. immediately upon discovery. Sampling in such cases is only to establish possible links f) Air sampling shows moulds of indoor between growth at these sites and airborne or origin are absent or below benchmark; no sedimented inoculum sampled elsewhere. Stachybotrys is present. Rely on #2, #3, and #5 (#6*) to detect any temporary and poorly disseminating amplifiers. 18

31 a) Initial procedures where symptoms are Estimate the number of moulds of indoor absent, analytical resources are limited, or origin, as gauged by species-by-species a preliminary survey is being conducted: comparison with outdoor controls. Species within air ducts and HVAC coils, etc., do need not be named in these comparisons, but #7 and #8*; visually inspect any isolates must be recognized as conspecific, functioning humidifier, aerosolizer, based on mycological analysis of macroscopic vaporizer, or mister; if water is turbid, take or microscopic characters. Identification of a sample for microscopic examination Aspergillus species, Penicillium subgenera, (#17) and culture (#18). and genera of other numerically significant fungi is recommended. Minor species present b) Examination for evidence of fungi in settled in proportions below 1% can be ignored materials: dust, biofilms, and standing provided any Stachybotrys, Aspergillus, or water. Rigorous procedure where other pathogenic or toxigenic fungi known to symptoms warrant investigation and be of concern are detected. resources permit: do all procedures outlined in #5, as well as #6. Certain Note that some workers sample dust by plating procedures currently under applied it directly on growth medium (often dichloran research investigation may become 18% glycerol medium, which favours the increasingly used in these investigations, growth of dust-associated xerophiles), since including fungal ergosterol or glucan making aqueous suspensions may be difficult. sampling (#15) or, in environments with Others prefer to gain an approximate measure mycotoxigenic species present or expected, of human exposure to settled dust by direct mycotoxin sampling (#16) or disturbing dusty materials and air sampling cytotoxicity testing (#19). with a volumetric technique. In water dilutions #6: Dust Vacuum Sampling and in air sampling of disturbed dust, clumps of hydrophobic spores or conidia may remain largely intact, yielding colonies that actually Collect floor or carpet dust using a suitable reflect agglomerations of potential colonyvacuum device; also compare with outdoor forming units. In dilutions where a wetting dust control*. On a subsample of bulk dust, agent (e.g., dimethyl sulphoxide) is used, they perform direct microscopic examination (#7); may break up and give a stronger appearance also do dilution series in sterile water or other of heavy infestation. appropriate diluent, beginning with a measured quantity, e.g., 1 g dust suspended in 10 ml a) If moulds of indoor origin are present at fluid (1:10 suspension), subsampling 1 ml of negligible levels or below the benchmark suspension to 9 ml fluid to make a 1:100 level (see below), take no further action suspension, and likewise 1:1000, 1:10 000, unless indicated by other procedures. The and 1: *. Plate 0.1 ml from each presence of any quantity of Stachybotrys suspension onto high water activity and low always warrants further investigation go water activity growth media as per procedure to #6b. Re benchmark: there are currently #4, with replication. Analyse CFU/g dust no proposed benchmarks for based on counts on the dilution that has more than 25 and fewer than 100 colonies per plate, or a number close to one of these figures. 19

32 such studies, but experimental studies circumstances) but either not found in possibly yielding such benchmarks for outdoor controls or found in outdoor different dust sampling techniques are in controls at statistically significantly lower progress. If available, please use. In the levels (chi-squared test). Go to #12. meantime, experienced evaluators may judge whether moulds are present at biv) Minimal Stachybotrys found indoors (see unusual levels suggestive of indoor #6bii for definition of minimal) and also amplification, or whether individual species in outdoor controls at minimal or higher or species associations suggestive of indoor level. Go to #13. proliferation are present. If so, go to #6b. bv) Low to high (see #6biii for definition) b) If moulds of indoor origin are present at numbers of Stachybotrys found indoors levels indicative of substantial indoor and also in outdoor controls at amplifiers or at levels above benchmark insignificantly different (chi-squared test) (see comment on benchmark under #6a, or higher levels. Go to #14. above) for total fungal count, or if individual species of concern or ecological #7: Microscopic Examination of Mould or categories (such as toxigenic fungi) of Dust concern are present, or if species composition of dust spora suggests indoor Mount dust sample, mould scrapings, proliferation, then go to the appropriate transparent tape sample, or surface scrapings subprocedure of #6b: on a microscope slide with a hydrophobicitybi) Moulds other than Stachybotrys are reducing mounting medium (e.g., water + Roccal or other laboratory detergent; 25% present indoors at levels considered to be sodium hydroxide; or ethanol followed of potential concern: go to #10. immediately by water) and examine under 10 bii) Minimal Stachybotrys found indoors but and 40 for characteristic structures of fungi, particularly fungi of known concern. In surface not found in outdoor controls. (Minimal scrapings, a site of active fungal growth, i.e., = a vanishingly small number detected: an amplifier, is recognized by the presence of for example, a single isolated colony hyphae and (in most cases) conidiophores as found under any circumstances, or two well as conidia of the same species. Other widely dispersed colonies found in a fungal sporulating structures such as ascomata large series of samples from different may also be present. Heavy deposits of conidia rooms. Note that colony means an or spores alone may signify either heavy actual fungal colony observed on the deposition from another source or an old, sampling medium after incubation, not a inactive amplifier in which structures of active CFU/g in calculation.) Go to #11. growth have broken down; alternatively, it may simply indicate that the surface sampled biii) Low to high numbers of Stachybotrys has not been abraded aggressively enough to colonies found ( low to high : at least detach hyphal structures from the substratum. three colonies found in a multi-room sample or two colonies from a single room; or more colonies found under any 20

33 Taking these and any other salient factors into e) Microscopic mount negative for fungal account, the experienced investigator should structures or nearly so. Take no further make a judgement about whether the material action unless another procedure (#3, #4, examined is from an amplifier. For general #5, #6*) indicates significant indoor mould remediation of mould amplifiers, see Section proliferation in other locations. 5.1 of this document. A specialized remediation protocol for Stachybotrys has #8: Culture of Swabs, Scrapings, or Surface 28 been formulated. Dust; and Use of Contact Culture Plates a) Microscopic mount shows Stachybotrys Surface dust culture, swabs, scrapings, or from amplifier site: if site is known, go to contact plates will all suffice to detect fungi on 28 Stachybotrys remediation protocol ; if surfaces but will not distinguish between unknown, or if more sites may exist, go to species growing on the surface and species 7c. that have merely been deposited there as inactive propagules. These techniques are b) Microscopic mount shows other mould substantially interchangeable, and their use from amplifier site: clean as warranted, should be tailored to the situation and the based on type and extent of mould (see sampling materials available. Section 5.1). Sufficiently large quantities of dust or other c) Microscopic mount shows no evidence of material that can be suspended more or less an amplifier but Stachybotrys conidia evenly in water can be cultured by dilution present, or mount shows evidence of a series as outlined in #6. Swabs should be Stachybotrys amplifier for which the streaked on high water activity and low water location cannot readily be traced (e.g., activity media (see #4) as per normal conidiophores from an unknown source microbiological inoculum attenuation present in bulk dust sample): rely on #2, procedure (e.g., limiting the original swab #3, #5, repeating or extending if necessary, streak to one-third of the plate and crossand, failing these, #9 or expert streaking with a sterile loop through three or consultation, to locate source. When the four partial rotations of the plate). Small source is found, go to Stachybotrys pieces from scrapings, particularly from 28 remediation protocol. mouldy-looking areas, should be suspended in sterile water; the suspension should be d) Microscopic mount shows no evidence of vigorously agitated or sonicated and plated out an amplifier, but significant levels of fungal directly on high water activity and low water material present, or mount shows evidence activity media. Contact plates should be of an amplifier for a fungus other than applied to surfaces where mould growth or Stachybotrys, but the exact location of this deposition is suspected and incubated at room amplifier is not apparent: rely on #2, #3, temperature (or, where invasive opportunistic #4*, #5 (extended if necessary), #6* to pathogens are specifically being sought, at assist in locating amplifier site(s). If an 37EC) for at least seven days. amplifier is located, go to remediation protocol (Section 5.1). 21

34 a) Culture shows Stachybotrys in scrapings, Section 5.1. Select additional sample sites swabs, or contact plates; to find source, in similar or nearby areas and proceed rely on #2, #3, #5, and #7, repeating or according to #5, #7, and #8. Reiterate until extending if necessary; failing these, rely on no more sites of heavy mould inoculum are #9a, #9b, or expert consultation. For detected. Stachybotrys in dust, proceed as per #6bii #6bv as appropriate, or, where c) Scraping, swab, contact plate, or surface outdoor controls are lacking, consider dust culture is heavily positive for bacteria proceeding as per #6*. If the original or yeast, with few or no moulds present. Stachybotrys in swabs, scrapings, or There is a very moist environment, likely contact plates was minimal, seen only as occurring indoors in conditions of high one or two colonies in a large sample, and humidity, flooding, or condensation, which if #9a and #9b fail to show evidence of will also produce mould in less moist Stachybotrys, then do a new #3, #4 (or habitats. Rely on #3, #4 (or #6*), and #5 to #6*), and #5. If these are again positive but detect the degree of mould growth and minimal, consult the outdoor controls from sites where it occurs. #4 or #6 and proceed as per #6bii or #6biv, whichever is more appropriate. If the d) Scraping, swab, contact plate, or surface repeats are again positive but levels rise dust culture is negative for mould or nearly beyond minimal, then consult the outdoor so. Take no further action unless a controls and proceed according to #6biii or previous protocol (#3, #4, #5, #6) has 6bv, whichever is more appropriate. If the indicated significant indoor mould repeats are negative, then disinfect the site proliferation in other locations. where Stachybotrys was isolated. If no symptoms are reported, take no further #9: Extraordinary Physical Search: action; if symptoms are reported and #9a Examination of Difficult Sites within has been done, consider doing #9. If #9 and Building all other procedures following the initial minimal isolation of Stachybotrys are Previous flooding may have resulted in negative but symptoms continue to be accumulations of mould conidia within wall or reported, consider monitoring again in 2 3 false ceiling cavities, in wall or duct insulation, months in addition to testing non-fungal on the backings of carpets, or in other potential causes of symptoms. inconvenient locations. Walls and false ceilings are sufficiently enclosed to serve as humid b) Scraping, swab, contact plate, or surface chambers promoting mould growth, yet they dust culture grows profuse toxigenic or are often sufficiently porous to allow the allergenic mould but not Stachybotrys: rely dissemination of conidia or toxic or antigenic on #7 to confirm growth at sampling site. If hyphal or conidial fragments. Actively growing #7 is positive, see instructions for #7. If #7 mould may produce offensive, volatile, odouris negative (e.g., if growth is derived from causing substances. Flooding history or the small, sedimented conidia obscured by results of air, dust, or surface samples may debris), rely on #2, #3, and #5 to locate the suggest site of growth and also disinfect the broad area around the positive sample site as per 22

35 that easily accessible sites are not growing general spore accumulation. More enough mould to explain the observed mould stringently, use a rigid endoscope levels or mould-related symptoms, and that a (borescope) as outlined in #9a or cut an possible cryptic mould reservoir exists. In such approximately 15 cm 15 cm hole in cases, difficult samples, requiring entry of representative wall sections and inspect ordinarily inaccessible spaces, must be visually. Perform 7, 8* on exposed undertaken. interiors, and in particular on suspected amplifiers. a) History (#2) or physical inspection (#3) of the site suggests previous or current bii) Ducts: Use existing access openings or a flooding in a poorly accessible site (e.g., rigid endoscope (borescope) to inspect wall cavity, false ceiling interior, wall or duct interiors. Perform #7 and #8* duct insulation, carpet backing): expose a adjacent to access hole or borehole. representative portion of the environment Where material suggestive of fungal (e.g., wall interior or carpet backing) in the amplifiers or spore deposition is seen at area of concern and perform #7 and #8* in a distance from original entry point, use the newly exposed environment. If elongated scraping device or drill available, a rigid endoscope (borescope) additional holes as necessary and perform may be used to examine cavity interiors #7 and #8*. Holes can be sealed with a with minimal damage; perform #7 and #8* plug. adjacent to borehole. Where material suggestive of fungal amplifiers or spore #10: Mould (Non-Stachybotrys) Source deposition is seen using endoscope, drill Location and Clean-up additional holes as necessary and perform #7 and #8*. Procedures such as air sampling and dust sampling do not directly disclose the location b) In sites where no historic information is of amplifiers (i.e., sites of indoor mould available and no water damage is recalled proliferation). If potentially problematic levels or seen but where investigation is still of moulds of indoor origin are revealed by warranted by symptoms or other concerns, these techniques, the sources of these mould perform one or more of the following tests propagules must be discovered. Stachybotrys with stringency appropriate for the degree is treated separately because a stringent cleanof health concern: 28 up protocol has been described. bi) Wall cavities: A low stringency test is to a) Find the source of (non-stachybotrys) check a few representative wall cavities mould: rely on #2, #3, and #5 to suggest behind switch face plates. Use an the location (follow any procedures alcohol-disinfected, flamed, bent wire to branching from #5); if necessary, perform scrape small amounts of material from extended #3 and #9a* or #9b*. When the back or front of wall cavity, being careful source is found, remediate (see Section to avoid electric wires, and perform #7 5.1). and #8*. This test is subject to false negative results if amplifiers are discontinuously distributed but can detect gross,confluent contamination or heavy 23

36 #11: Minimal Stachybotrys, Source Not One or two colonies of Stachybotrys in an Located, Likele Indoors indoor survey may be the viable representatives of large numbers of non-viable Stachybotrys occurs on cellulose that has propagules, thus indicating a serious level of become moist. Although it may occur indoors contamination. Alternatively, they may reflect in large quantities on structural materials and fortuitous isolation from a minor source. In bulk stored materials, it may also occur either case, repeated air or dust samples sporadically on small cellulosic substrata or in analysed by culture may yield a false negative low abundance in marginal habitats. For result. The best procedure to use as a followexample, a library may contain a very low up to positive cultures is a search for possible number of previously water-damaged books Stachybotrys sources. Only when this search that may still shed a few Stachybotrys conidia. gives a negative result is it reasonable to A few Stachybotrys isolates in an air or dust attempt repeat culture samples. This is done to survey may derive from such origins. exploit the possibility that, if significant Likewise, dust of outdoor origin may serve as amounts of Stachybotrys are present, they may a reservoir for a small number of Stachybotrys recur at least some of the time in culture conidia indoors, and one or two colonies may samples. In other words, when better turn up on indoor surveys even when techniques have failed, this imperfect contemporaneous outdoor control samples are technique may be used because of the chance negative. that it may yield valuable information. The isolation of a small number of a) Rely on #2, #3, and #5 (and, failing these, Stachybotrys colonies is difficult to interpret. #6*) to suggest the site of Stachybotrys One of the attributes rendering Stachybotrys growth. If #3 is negative and #5 or #6 is distinctive is the low viability of its conidia positive for visible conidia or culturable encountered in indoor situations. Hundreds of propagules, perform an extended #3 and conidia seen in a direct examination may show #9*; if all tests are negative, repeat #4 or only 2 3% viability in culture. Results from do #6 (repeat #6 if it was the original using only culture techniques alone may source of Stachybotrys isolation) in the profoundly underestimate the presence of this area where Stachybotrys was detected. If organism and therefore its toxic effect. Dead the repeat test is negative, disregard conidia apparently remain toxic for some time, Stachybotrys; if the repeated results of as do conidial fragments. Undoubtedly the either #4 or #6 are positive but minimal (a decline in conidium viability occurs over time, single colony or two widely dispersed and most or all conidia are likely viable when colonies in a large series of samples), rely they are fresh. Yet Stachybotrys material on direct microscopy of dust as per #7 to investigated by indoor mould researchers tends determine if dead Stachybotrys conidia are to show low culture viability. It may be that present; also perform #9* or #9a if not this highly toxic material is less likely than performed already; if no Stachybotrys other environmental contaminants to be conidia or sources are evident, disregard degraded into an inoffensive form after it has Stachybotrys. If conidia are evident in #7, lost viability. do an extended #3 and #9; if no source is found in these procedures, call 24

37 an expert. If #6 is positive for low (three or a) If a low-level or distant outdoor more colonies overall or two clustered Stachybotrys source is possible, rely on #2, colonies; or a single colony in a small #3, and #5 (and, if these fail, #6, repeated sample) to high, follow procedure #12 if if done before) to clarify indoor levels and outdoor controls are negative or #14 if sources. If new outdoor controls and #2, outdoor controls are positive. #3, and #5 are negative and a new indoor dust sample (#6) is positive but minimal (a #12: Low to High Stachybotrys, Source Not single colony or two widely dispersed Located, Likely Indoors colonies in a large series of samples), consider monitoring on one or more further The finding of low to high Stachybotrys occasions; if the outdoor controls are colonies strongly implies that an indoor negative but indoor levels are low (three or amplifier is likely to exist. In such cases, it is more colonies overall or two clustered necessary to find the amplifier. colonies; or a single colony in a small sample) to high, the likelihood of an indoor a) Rely on #2, #3, and #5 (and, if these fail, source is heightened; follow #12. If the repeat #6 if it was done only once before) outdoor control is positive but the indoor to suggest the site of Stachybotrys growth; control is negative, disregard indoor if the source is found, remediate as per Stachybotrys procedures (if the outdoor 28 Stachybotrys remediation protocol ; if the source yields high levels, consider source is not found, perform an extended searching for and eliminating it). If both #3 and #9 or #9b. If all tests are negative, outdoor and indoor samples are negative, repeat #4 or do #6 (repeat #6 only if it was discontinue Stachybotrys procedures. done once before; if you already have a result of a repeat trial, rely on that); if the #14: Low to High Stachybotrys, Source Not repeat #4 or #6 is negative, consider Located, Outdoors or Indoors monitoring on one or more further occasions or, where symptoms are of See comments under #13 on possible concern, consult an expert; if the repeat #4 occurrence of Stachybotrys in the outdoor or #6 is positive, further extend #2, #3, #5, environment. and #9 exhaustively or consult an expert. a) Controls of previous studies have indicated #13: Minimal Stachybotrys, Source Not that a nearby or relatively high level Located, Outdoors or Indoors outdoor source of Stachybotrys is possible. Nonetheless, an indoor source also remains See comments under #11 on the significance possible where conducive habitat of minimal numbers of Stachybotrys colonies. conditions exist or have existed; an Note that Stachybotrys is common in certain outdoor amplifier may have served as an agricultural situations, such as decaying straw, inoculum source for indoor colonization. especially in horse barns. Stachybotrys may Rely on #2, #3, #5, and #9b* to detect any also occur on other natural moist cellulose indoor amplifier; if the original substrata and hence may appear in outdoor Stachybotrys-positive controls in #4 or #6 control samples. were within 6 m of the test 25

38 building or downwind of possible air established protocol for interpreting $-1,3- outflow from the building, repeat #4 or do glucan results from allegedly mould- #6 (repeat #6 only if done only once contaminated buildings does not yet exist but before; if you already have a result of a is an area of active research. repeat trial, rely on that) with outdoor controls in more distant, windward (#4) or #16: Mycotoxin Sampling from Air or Dust normally windward (#6) sites. If new outdoor controls are negative and indoor In certain cases, significant levels of a controls are positive for Stachybotrys at particular mycotoxin may be expected to occur any level, go to #12. If outdoor levels in in an environment, either because of high the repeat still differ insignificantly (chi- occurrence of the associated mould or because square) from or exceed indoor levels, aerosols deriving from a substrate for discontinue indoor analysis or, if symptoms toxigenic mould growth (e.g., peanut or corn are of concern, consult with an expert. If dust in the case of Aspergillus flavus) are new outdoor controls are positive for suspected to be present. Direct mycotoxin Stachybotrys but indoor levels are detection mechanisms ranging from simple significantly greater (chi-square), then go medium tests to complex chromatography and to #12. If new indoor and outdoor tests are spectrometry may be used to determine both negative for Stachybotrys, discontinue mycotoxin levels. A large literature exists on analysis. analytical techniques for various mycotoxin classes, but review of this literature is beyond #15: Ergosterol or Glucan Sampling the scope of this document. Ergosterol is a cell membrane component In many contaminated indoor environments, (analogous to human cholesterol) that is the substances responsible for causing specific to fungi. Quantitative analysis of symptoms are not well known and cannot be ergosterol in an environment can be used to detected directly. Most moulds produce estimate the level of fungal contamination complex mixtures of mycotoxins and may present. It cannot identify whether this produce toxic proteins and irritating antigens contamination derives from indoor amplifiers, in addition to the classic small mycotoxin sedimentation of outdoor airspora, or fungi in molecules. Hence, direct mycotoxin imported mud and debris (e.g., from shoes, monitoring is applicable only where there is a dog or cat feet). Nonetheless, markedly high clear environmental dominance of an individual indoor levels generally indicate problematic toxin-producing species or a special concern fungal amplifiers, and this inference is easily regarding a particular toxin or class of toxins. confirmed with species-by-species analysis of An example of such a concern is the possibility a small number of air or dust samples. For of exposure to aflatoxins, predisposing 13 further information, see Miller et al. workers to liver cancer, in a peanut processing factory where symptoms and adverse health $-1,3-glucan is another fungal biochemical, a effects suggest exposure to mycotoxins. cell wall component, that may be measured in #17: Humidifier Water Direct Microscopy an attempt to assess the total quantity of viable and non-viable fungal biomass. A well- Water from humidifiers and misters may 26

39 contain profuse growth of micro-organisms, should not be necessary, as the contamination including moulds and yeasts, as well as is dense in significantly contaminated bacteria and protozoans. Clean humidifier humidifiers. Indeed, a dilution series (as in #6, water should appear clear. If it does not, the simply dilute aqueous suspensions 1:10, 1:100, cause of turbidity may be biological or and so on) may be desirable to obtain wellchemical. Chemical turbidity usually consists separated, countable colonies that are of a sediment of calcium carbonate crystals; in relatively free of overgrowth by antibioticareas where water is high in calcium, this resistant bacteria. Sampling of bacteria and sediment may heavily encrust evaporation- endotoxin should also be considered. based humidifiers. Chemical turbidity arising from the suspension of fine carbonate particles #19: Cytotoxicity Testing may be difficult to distinguish from biological turbidity. Microscopy and culture may be used Dust collected by means of a vacuum device to make this distinction. may be tested directly for toxic components without targeting particular toxins. A Water should be taken in a sterile container. technique for accomplishing this utilizes a Mounts should be made and examined sensitive cultured human cell line and exposes microscopically, looking for distinctive fungal it to chemical extracts of the dust. filaments, yeast cells, and conidia. Centrifugation may be used to concentrate the 13 Miller et al. adapted a technique employing biological matter for examination. human HeLa cells for dust cytotoxicity studies; the reader is referred to that publication for #18: Humidifier Water Culture further information. Water should be plated directly onto any Cytotoxicity analysis detects overall levels of general fungal growth medium (ideally, media toxins in dust, not just mycotoxins, and the restricting colony diameter such as Littman relative importance of mycotoxins in known oxgall agar, rose bengal agar, or inhibitory cytotoxic samples must be inferred from fungal mould agar) containing antibacterial analysis (culture and/or direct microscopy) of antibiotics. Prior centrifugation the same samples. 27

40

41 V. Remediation and Preventive Maintenance of Buildings 5.1 Remediation should be in place for disposal of contaminated materials (see Appendix C). Strategies for the remediation of indoor air Duct cleaning is frequently accomplished quality problems caused by fungi are based on by vacuuming contaminated surfaces. the elimination of conditions that promote the Conventional vacuuming equipment should not amplification of these potentially hazardous be used in occupied areas because of the organisms. Typically, amplification occurs in possibility of transferring the contaminants into environments where excess water is the air. Only vacuuming devices equipped with 2,33 available. This situation is commonly found HEPA filters should be used if the cleaning is in areas that have been flooded or where 28 done inside occupied space. In all cases condensation has taken place. Air ducts in where remediation has been accomplished, it which moisture has collected, dampened should be followed up with routine cleaning ceiling tiles, wall panels, carpets, and and maintenance. insulation also provide ideal conditions for the In some cases, cleaning is not possible growth of fungi. Remediation of fungal because of the nature of the contaminated hazards involves cleaning, removal of material. Carpets, insulation, and porous contaminated materials, and modification of ceiling or wall panels fall into this category. affected environments. When these materials become contaminated, Clean-up of fungal contaminants associated there is no way to determine if fungal growth with hard or non-porous surfaces such as has been eliminated by cleaning, and complete walls, air ducts, cooling coils, and drain pans removal of the affected materials is therefore that collect water should be carried out when necessary. Precautions taken during removal 28 the affected area is unoccupied. Trained of contaminated materials should be similar to clean-up personnel should use appropriate 28 those followed for cleaning activities. For protective equipment such as respirators and example, appropriate protective equipment gloves during cleaning activities (see Appendix should be used, and removal of contaminated C). The use of biocides for clean-up of fungal material should be done by trained individuals. contaminants is usually discouraged because of As the removal of porous contaminated the potential toxic effects for cleaning materials may result in the creation of personnel and other individuals who may be hazardous aerosols, it may be necessary to exposed. However, if well-characterized isolate the area with plastic sheeting and carry biocides are used according to manufacturers' out the remediation procedure under negative specifications, and if they are properly applied, pressure. This precaution prevents the they may provide a valuable adjunct to transport of aerosols to non-contaminated 2 cleaning procedures. The use of household areas (see Appendix C). Furthermore, bleach has been suggested for contaminated following removal of the surfaces. Following clean-up, appropriate procedures 29

42 contaminants, it is good practice to disinfect eliminating future indoor air quality problems. hard surfaces in the containment area with Although remediation is necessary when chlorine bleach and to vacuum the area with a fungal hazards have been identified, a much HEPA filter-equipped vacuum cleaner. One of better strategy is to prevent the occurrence of the more important aspects of this type of 36,37 problems. Contamination can be prevented remediation is the proper disposal of by thorough routine cleaning and maintenance contaminated materials. This would include and by following some of the modification appropriate containment in sealed plastic bags strategies indicated above. Prevention of during transport to a landfill disposal site. If fungal contamination is one of the most disinfection or sterilization is necessary, this desirable strategies for risk management. can be accomplished with biocides or by incineration or high-pressure steam in an autoclave. The replacement of contaminated materials should be done so as to prevent the 5.2 Preventive Maintenance The design, construction, and maintenance recurrence of the contaminants by eliminating of public buildings should minimize conditions those conditions that led to their original that allow the accumulation, amplification, and amplification. dissemination of micro-organisms in indoor Many of the indoor air problems associated air. A wide-ranging discussion on building with fungi can be remedied simply by preventive maintenance is beyond the scope of modifying the environment where the this document, but a few general principles 35 problems occur. For instance, moulds do not should be considered in the development of require standing water to grow. High relative programs to avoid the development of fungal humidity (RH) can provide sufficient moisture amplification sites. Detailed instructions on for growth on the surface of porous or non- preventive maintenance to control fungal porous materials if the surface temperature is 36,37 growth in public buildings are available. below the dew point. Fungal amplification might be eliminated under these conditions by Background simply raising the temperature or by The foregoing sections have dealt with dehumidifying those areas known to promote the recognition and the correction of fungal growth. Leaky water pipes linked to fungal problems. The context has been that fungi growth should be repaired. The absence of should not be present above acceptable levels. available moisture may be sufficient to prevent Elevated levels of fungi are generally indicative fungal amplification. In practice, removal of of amplification sites or poor outside air water sources should prevent the growth of filtration. This state of affairs denotes a fungi. maintenance problem that must be located and Preventing condensation on walls or other alleviated, using the strategies outlined. surfaces can be accomplished by redirecting air However, these can be considered secondary 28 flow to eliminate cold spots. Condensation strategies, as the primary aim should be to points can also be eliminated by the construct buildings that have been designed to appropriate application of a vapour barrier or avoid the development of insulation. Moisture control is the best strategy for controlling fungal growth. Therefore, modifying the environment to prevent moisture is the best approach for remediation and for 30

43 fungal problems. Also, preventive maintenance Areas of potential water condensation on cold programs must be defined and regularly surfaces such as external walls, water pipes, implemented in existing buildings, with the and ducts can be eliminated by proper objective of safeguarding against the insulation, ventilation, and humidity control. appearance of fungal problems. Building maintenance personnel and 3. Maintaining a sufficient humidity level: building managers should be aware of the Humidity should be regulated at a sufficient potential health problems associated with level, high enough for the comfort of the contaminated indoor air, including the occupants but not so high as to promote importance of the proper design, installation, condensation. These criteria are generally operation, and maintenance of HVAC systems considered to be satisfied at relative to minimize the accumulation, amplification, 38 humidities in the range of 20 60%. When and dissemination of micro-organisms. Staff establishing RH levels in buildings, the responsible for operating the building systems procedure should follow good industrial should receive intensive training in all hygiene practices and occupational health applicable maintenance procedures. The and safety legislation and regulations in the absence of such knowledgeable staff members area of jurisdiction. and the low priority often allotted to the implementation of maintenance programs are 4. Facilitating preventive maintenance: Sites among the most common factors that lead to of known and potential water accumulation the appearance of fungal and other should be constructed to readily allow environmental problems in buildings. Use of inspection and service. steam is preferred over use of water spray for humidification Implementation of a Preventive Maintenance Program Building Design Considerations The building operating systems include Several aspects of building design can be all components of the heating, cooling, and implemented to minimize the amplification of humidification systems and the air handling fungal contaminants in indoor air: and distribution units. The preventive maintenance program should be directed 1. Limiting access of the outdoor aerosol: towards minimizing fungal amplification sites These considerations minimize the entry of by ensuring adequate drainage of sumps and outdoor fungi into the building air, through drip pans, regular cleaning of dirt and slime the provision of a tight structural envelope, from all constituents, and replacement of of particle filtration of the intake air, and of filters. The frequency of conducting these climate control to minimize the need for procedures varies with each component, from opening windows. monthly to annually. Porous lining materials should not be present in any part of the HVAC 2. Eliminating sites of water accumulation: system. Sites of unavoidable water collection in cooling and humidification systems should be constructed to be completely drained. 31

44 The building constituents include all importance of the proper design, installation, components of the building envelope and operation, and maintenance of HVAC systems interior. Any sources of external and internal to minimize accumulation, amplification, and leaks and condensation should be promptly dissemination of micro-organisms. and permanently corrected. Water-damaged Information-sharing networks should be insulation, ceiling and wall materials, carpets, established to assist public health officials and upholstery, and other porous components may others concerned about the microbiological need to be removed. quality of indoor air to quickly and effectively deal with problems in public buildings. Public Communications education to increase awareness of the Building maintenance personnel and possible contribution of microbiologically building managers should be aware of the contaminated indoor air to allergenic, toxic, or potential health problems associated with infectious illnesses is essential. contaminated indoor air, including the 32

45 VI. Recommendations for Future Actions and Research During the course of its deliberations, data on fungal spores and other propagules, the Working Group on Mycological Air mycotoxins, and other volatile metabolic Quality in Public Buildings identified the compounds (e.g., ergosterol and ß-1,3- following issues that should be addressed glucan). before health-based guidelines for the mycological quality of indoor air of public 3. Health Effects Assessment buildings can be developed: Dose response data on quantities of 1. Detection Methods for Exposure airborne fungal propagules, toxins, and volatile Assessment compounds required to initiate infection, illness, or allergenic response are required. Sampling protocols and analytical methods This information, along with epidemiological for fungi should be standardized. Analytical studies, is essential to establish meaningful methods based on immunological (e.g., health-based mycological guidelines for public monoclonal antibodies) and molecular (e.g., buildings. Health Canada, the Canada polymerase chain reaction and gene probes) Mortgage and Housing Corporation, and techniques should be developed. Standard Agriculture and Agri-Food Canada are methods for the measurement of mycotoxins currently conducting such a study. and assessment of their toxicity should be developed, including in vitro screening using 4. Interactive Computer Model various biological indicators. The limitations and biases of the selected methods must be The development of an expert system, well recognized. based on the Protocols for Investigation of Studies indicate that allergenic effects Indoor Fungal Amplifiers (see Section 4.4), cannot always be correlated with the presence should be considered to simplify the of fungi in indoor air. The use of allergen investigation and interpretation of indoor extract standards, prepared from fungi fungal contamination. associated with indoor amplification, should be The Working Group also strongly explored to determine if hypersensitivity recommends that the Federal-Provincial reactions can be related to the presence of Committee on Environmental and these fungi in indoor air. Occupational Health consider the establishment of new working groups to 2. Reference Values for Exposure address, individually, other indoor Assessment microbiological contaminants of concern to public health, such as viruses, bacteria Using standard sampling protocols and (including mycobacteria and actinomycetes), mycological methods, bioaerosol data from protozoans, and dust mites. public buildings and adjacent outdoor locations in all regions of the country should be collected and published. These should include baseline quantitative and qualitative 33

46 VII. References 1. U.S. Department of Labour, 6. Jarvis, B.B. Mycotoxins and indoor air Occupational Safety and Health quality. In: Biological contaminants in Administration. OSHA indoor air indoor environments. P.R. Morey, J.C. quality proposed rule. 29 CFR Feeley, Jr., and J.A. Otten (eds.). Parts 1910, 1915, 1926, and 1928 ASTM Spec. Tech. Publ. 1071, (April 5, 1994). Washington, DC. p. American Society for Testing and (1994). Materials Committee D-22 on Sampling and Analysis of 2. Health Canada. Indoor air quality in Atmospheres, Section 06 on Biological office buildings: a technical guide. A Aerosols (1990). report of the Federal Provincial Advisory Committee on Environmental 7. Tobin, R.S., Baranowski, E., Gilman, and Occupational Health. 55 pp. A., Kuiper-Goodman, T., Miller, J.D., (1993). and Giddings, M. Significance of fungi in indoor air: report from a working 3. Broder, I. Primer on airborne fungi group. Can. J. Public Health, 8 and other microorganisms for safety (Suppl.): 51 (1987). officers of Human Resources Development Labour Component. 8. Croft, W.A., Jarvis, B.B., and Final report to Technical Services Yatawara, C.S. Airborne outbreak of Division, Occupational Safety and trichothecene toxicosis. Atmos. Health Branch, Labour Component, Environ., 20: 549 (1986). Human Resources Development, Government of Canada, 29 March 9. Hodges, G.R., Fink, J.N., and (1994). Contract: 1993, File YR828- Schlueter, D.P. Hypersensitivity pneumonitis caused by a contaminated cool-mist vaporizer. Ann. Intern. 4. Burge, H. Bioaerosols: prevalence and Med., 80: 501 (1974). health effects in the indoor environment. J. Allergy Clin. 10. Salvaggio, J. and Aukrust, L. Mold- Immunol., 86: 687 (1990). induced asthma. J. Allergy Clin. Immunol., 68: 327 (1981). 5. Flannigan, B., McCabe, E.M., and McGarry, F. Allergic and toxigenic micro-organisms in houses. J. Appl. Bacteriol., 70 (Suppl.): 618 (1991). 34

47 11. Sorenson, W.G., Frazer, D.G., Jarvis, 17. Yoshida, K., Ando, M., and Araki, S. B.B., Simpson, J., and Robinson, V.A. Acute pulmonary edema in a Trichothecene mycotoxins in storehouse of moldy oranges: a severe aerosolized conidia of Stachybotrys case of the organic dust toxic atra. Appl. Environ. Microbiol., 53: syndrome. Arch. Environ. Health, 44: 1370 (1988). 382 (1989). 12. Tarlo, S.M., Fradkin, A., and Tobin, 18. Martin, C.J., Platt, S.D., and Hunt, R.S. Skin testing with extracts of S.M. Housing conditions and ill health. fungal species derived from the homes Br. Med. J., 294: 1125 (1987). of allergy clinic patients in Toronto, Canada. Clin. Allergy, 18: 45 (1988). 19. Platt, S.D., Martin, C.J., Hunt, S.M., 13. Miller, J.D., Laflamme, A.M., Sobol, and Lewis, C.W. Damp housing, mold growth, and symptomatic health state. Y., Lafontaine, P., and Greenhalgh, R. Br. Med. J., 298: 1673 (1989). Fungi and fungal products in some Canadian houses. Int. Biodeterior. 20. Rylander, R., Persson, K., Goto, H., Bull., 24: 103 (1988). Yuasa, K., and Tanaka, S. Airborne beta-1,3-glucan may be related to 14. Smoragiewicz, W., Cossette, B., symptoms in sick buildings. Indoor Boutard, A., and Krystyniak, K. Environ., 1: 263 (1992). Trichothecene mycotoxins in the dust of ventilation systems in office 21. Dales, R., Burnett, R., and buildings. Int. Arch. Occup. Environ. Zwanenburg, H. Adverse health effects Health, 65: 113 (1993). in adults exposed to home dampness 15. Di Paolo, N., Guarnieri, A., Loi, F., and molds. Am. Rev. Respir. Dis., 143: 505 (1991). Sacchi, G., Mangiarotti, A.M., and Di Paolo, M. Acute renal failure from 22. Dales, R.E., Zwanenburg, H., Burnett, inhalation of mycotoxins. Nephron, 64: R., and Franklin, C.A. Respiratory 621 (1993). health effects of home dampness and molds among Canadian children. Am. 16. Gordon, K.E., Masotti, R.E., and J. Epidemiol., 134: 196 (1991). Waddell, W.R. Tremorgenic encephalopathy: a role of mycotoxins in the production of CNS disease in humans. Can. J. Neurol. Sci., 20: 237 (1993). 23. Broder, I., Pilger, C., and Corey, P. Influence of volatile organic compounds and other environmental 35

48 variables on the well-being of workers Health Clinical Center. Guidelines on in office buildings. Report to National assessment and remediation of Health Research Development Stachybotrys atra in indoor Program, Health Canada, 31 March environments. New York, NY (1993). (1993). 29. American Society for Heating, 24. Skov, P., Valbjo/rn, O., Pedersen, Refrigerating and Air-Conditioning B.V., and the Danish Indoor Climate Engineers. ASHRAE Standard 62- Study Group. Influence of personal Ventilation for acceptable characteristics, job-related factors and indoor air quality. Atlanta, GA (1989). psychosocial factors on the sick building syndrome. Scand. J. Work 30. American Conference of Governmental Environ. Health, 15: 286 (1989). Industrial Hygienists threshold limit values for chemical 25. American Conference of Governmental substances and physical agents and Industrial Hygienists. Guidelines for biological exposure indices. Cincinnati, the assessment of bioaerosols in the OH (1993). indoor environment. Cincinnati, OH (1989). 31. Health Canada. Health risk communication handbook. Health 26. Wanner, H.-U., Verhoeff, A.P., Protection Branch (1994). Colombi, A., Flannigan, B., Gravesen, S., Mouilleseaux, A., Nevalainen, A., 32. Morey, P.R. Microbiological Papadakis, J., and Seidel, K. Indoor air contamination in buildings: precautions quality and its impact on man. Report during remediation activities. In: No. 12. Biological particles in indoor Environments for people, Proceedings environments. Commission of the of the ASHRAE IAQ '92 Conference, European Communities, Brussels Atlanta, GA. p. 94 (1992). (1993). 33. Canada Mortgage and Housing 27. Yang, C.S., Hung, L.-L., Lewis, F.A., Corporation. Moisture and air: and Zampiello, F.A. Airborne fungal problems and remedies. Householder's populations in non-residential buildings guide (1989). in the United States. In: Indoor air '93, Proceedings of the 6th International 34. Gravesen, S., Frisvad, J.C., and Conference on Indoor Air Quality and Samson, R.A. Microfungi. Climate. Vol. 4. Particles, microbes, Munksgaard, Copenhagen (1994). radon. P. Kalliokoski, M. Jantunen, and O. Seppänen (eds.). Helsinki, Finland. p. 219 (1993). 28. New York City Department of Health, New York City Human Resources Administration, and Mount Sinai Irving J. Selikoff Occupational 36

49 35. U.S. Environmental Protection 37. Nathanson, T. Microbial analysis in Agency/U.S. Department of Health office buildings. Technical Note, and Human Services. Building air Public Works and Government quality a guide for building owners Services Canada (1994). and facilities managers (1991). 38. Public Works Canada. Environmental 36. World Health Organization, Regional standards for office accommodations, Office for Europe. Indoor air quality: MD-15000, Section 3.1, November biological contaminants. WHO (1994). Regional Publications, European Series No. 31, Copenhagen (1988). 37

50 Glossary ACGIH American Conference of Governmental Industrial Hygienists. Actinomycetes Slow-growing branching filamentous gram-positive procaryotic organisms. Some thermophilic species may cause respiratory problems or allergic pneumonitis. Allergen An agent that induces an allergic reaction. Allergy A type of sensitivity to chemical, physical, or biological agents. Amplification The process of indoor growth leading to an increased indoor fungal concentration compared with the immediate outdoor environment. Amplifier An indoor substrate where fungal growth is occurring. Andersen sampler A sieve-type air sampling device that uses a vacuum pump to draw air through a radial pattern of 300 small pores, impacting particles in each of the small streams of air onto the surface of microbial growth medium. Andersen samplers with two or six stages separate particles by size and deposit each size class onto a separate plate. wall protein or glycoprotein, that is a constituent of one organism and is recognized and attacked by the immune system of another organism. The exposed or invaded organism forms antibodies that bind to the antigens of the circumambient or invading organism. ASHRAE American Society of Heating, Refrigerating and Air-Conditioning Engineers. Aspergillus A genus of mould fungi containing over 100 species, approximately 15 of which are commonly encountered in Canadian dwellings. All naturally occurring aspergilli are toxigenic. Aspergillus fumigatus A fast-growing thermotolerant mould that may cause opportunistic infection in immunocompromised persons or long-time asthmatics. Toxigenic and allergenic, it may also cause hypersensitivity pneumonitis in chronically heavily exposed individuals and non-specific respiratory symptoms in moderately exposed individuals. It is innocuous to the immunocompetent person under conditions of low exposure. Its habitats are compost, dung, and other organic matter, especially in warm areas with relatively high nitrogen content. Antigen A complex chemical substance, such as a cell 38

51 ß-1,3-glucan BRI A constituent of fungal cell wall suggested as Building-related illness. Recognized diseases one of the possible causative agents of adverse with a defined pathophysiology that can be effects in buildings with a history of water attributed to airborne building bioaerosols or damage. chemical pollutants (e.g., Legionnaire's disease, systemic mycoses, carbon monoxide poisoning, lung cancer). Bacterium (pl. Bacteria) A procaryotic organism, typically single-celled. Some of them can have pathogenic potential, others may cause problems in indoor air quality, especially when their mode of transmission is through the Building envelope The outermost enclosure element of a building. CFU respiratory route (e.g., Mycobacterium Colony-forming units. Small units of biological tuberculosis, Legionella pneumophila). material (spores, conidia, hyphal fragments) capable of giving rise to individual colonies on growth medium. Bioaerosol An airborne dispersion of particles containing whole or parts of biological entities, such as Conidia bacteria, viruses, rickettsia, protozoans, A term referring to the asexual spores of most actinomycetes, fungi, fecal elements or body types of mould (Hyphomycetes and parts of dust mites and other arthropods such Coelomycetes). These are much more common as cockroaches, and animal fur, skin scales, in air than are the more specialized sexual dander, hair, saliva, and urine. spores released by some of the same mould organisms. Biocide A chemical agent that kills a biological entity. Conidiophore Some common biocides are sodium The specialized branch producing the conidia hypochlorite, quaternary ammonium of a mould fungus. Seeing conidiophores in compounds, formaldehyde, hydrogen direct microscopy of indoor materials indicates peroxide, alcohols, phenolics, and the mould is actually growing and reproducing glutaraldehydes. in the site sampled. Biofilm A thin layer of micro-organisms growing across moist surfaces such as moist duct interiors and producing an adherent organic matrix. 39

52 Contact plate Direct slide A plate of microbiological growth medium A slide for microscopic analyses: prepared (e.g., fungal or bacterial medium) designed to from materials removed directly from be pressed directly onto a surface in order to contaminated surfaces without being subjected detect microbial inoculum, which, ideally, will to interim culture or other amplification grow on the medium in proportion to the techniques. degree of surface contamination. Dust mites Containment A safe condition brought about by effecting a Microscopic arthropod species common in the indoor environment. An important indoor barrier to microbial transmission. A allergen (e.g., Dermatophagoides containment device may consist of a stand- pteronyssinus, Glycophagus spp.). alone piece of equipment, a facility within a building, or a whole building that has the necessary engineering controls to keep Endotoxin A lipopolysaccharide (LPS) component of the biohazardous agents from escape. membrane of gram-negative bacteria and algae that is heat stable (resists autoclave conditions) Cryptococcus neoformans A pathogenic yeast growing in accumulated and toxic. A secreted toxin will be an exotoxin. bird (usually pigeon) or bat dung and causing cryptococcosis in heavily exposed or Ergosterol immunocompromised individuals. A membrane sterol specific for most fungi and Culture Cultivation of a micro-organism in a confined vessel: a technique employed to allow the sample material to multiply in number in order to facilitate its identification and assessment. Dimorphic systemic fungal pathogen One of a small number of specialized, virulent fungal pathogens growing in a mould form at room temperature and as a particulate phase (budding yeast, fission yeast, or spherule) at 37EC in the host or on specialized growth media. Blastomyces dermatitidis, causal agent of blastomycosis, and Histoplasma capsulatum, agent of histoplasmosis, are native to Canada. not significantly produced by higher animals or plants. FEV1 Forced expiratory volume in one second. The volume of air expelled in one second during a forced expiration from a full inspiration. Filamentous A tubular, apically extending, branched growth form characteristic of certain micro-organisms. Fungal spores In vernacular usage, differentiated fungal 40

53 reproductive structures such as conidia, chlamydospores, ascospores, etc., which facilitate dissemination and propagation. These are generally more resistant to adverse conditions than their corresponding vegetative state. A more restrictive definition of the term is adhered to in mycological technical literature: it refers only to propagules formed by a process of free cell formation, i.e., from a nucleus initiating an entirely new cell wall from cytoplasmic material. Hypersensitivity pneumonitis A chronic respiratory distress syndrome characterized by a type III (delayed type) allergic response to an immunosensitizing substance. Usually prolonged, heavy exposure to the allergic stimulus precedes development of the syndrome in susceptible individuals. Other equally heavily exposed individuals may fail to develop the syndrome, despite manifesting a strong immunological response to the prevalent antigens. Fungus Hypha (pl. Fungi) A kingdom of organisms (equal in (pl. Hyphae) A branching tubular structure rank to the Plant Kingdom or the Animal that forms the vegetative body of a growing Kingdom), defined technically as a parasitic or filamentous fungus. saprobic, filamentous or single-celled eucaryotic organism, devoid of chlorophyll and Hypochlorite characterized by heterotrophic growth, The ion that is the active ingredient in bleach, production of extracellular enzymes, and a! HOCl. It is a powerful antifungal disinfectant distinctive L-lysine biosynthesis pathway. but also bleaches many dyes and strongly Fungi (e.g., moulds, yeasts, mushrooms) may corrodes metal. cause indoor air quality problems through the dissemination of conidia, spores, toxins, or cell wall constituents. IgE Immunoglobulin type E are antibodies that mediate certain acute allergic reactions. HEPA High-efficiency particulate air filters that have been tested to assure removal of 99.97% of Impactor A sampling instrument employing impactor particles 0.3 Fm in size. plates to collect inspirable, thoracic, or respirable particulate matter using the principle HVAC Heating, ventilation, and air conditioning. that different-sized particles have different acceleration and removal rates. The impactor plates are located downstream of a sharp bend just below sharp edge slits. 41

54 MMEF Maximum mid-expiratory flow rate. The mean rate of expiratory air flow between 25 and 75% of the forced expiratory vital capacity. Monoclonal antibody An antibody secreted by a single clone of antibody-producing cells. Such antibodies have the same combining site, the same light chain, and the same immunoglobulin class, subclass, and allotype. Mould (American spelling: mold) Normally refers to fungi with a filamentous growth form, often giving rise to fuzzy, cottony, woolly, or powdery textured colonies. Moulds produce conidia or spores that are poorly visible or not visible at all to the naked eye and that in many species are specialized to become airborne. Mycelium Mass of hyphae derived either from a single fungal colony or from a group of associated fungal colonies. moniliformin; T-2 toxin, a Fusarium trichothecene). OH&S Act Provincial occupational health & safety acts and the Canada Labour Code for Federal Workplaces, which demand that the employers ensure that the workplace is safe for the employees and that the workers ensure the same for fellow workers and building occupants. Polymerase chain reaction A technique for amplifying material from a small amount of DNA segments using denaturation, annealing to primers, and DNA polymerase-directed DNA synthesis. Propagule Any disseminable fungal element that can give rise to a new fungal growth. Public buildings Offices, schools, etc., but excluding hospitals and industrial workplaces. Mycotoxigenic RCS Producing mycotoxins, specialized fungal Reuter Centrifugal Sampler. A centrifugal air toxins. Many different types of toxins are impaction device for quantitatively sampling included in this broad category. Most are microbial propagules. It uses an impeller fan to small, non-volatile molecules such as impact propagules onto the agar-filled wells of polyketides, amino acid derivatives (e.g., a moulded plastic strip lining the inside of the penicillin), alkaloids, trichothecenes, and so sampler. It is highly portable and hence may be on. The production of mycotoxins is usually easier to use than other equivalent stressed in indoor air mycology only when vacuum/culture devices. Blank strips can be toxins known to adversely affect humans and purchased to accommodate any special other mammals are produced. Each individual mycological media as required. Some newer toxin has its own spectrum of noxious effects, models allow for quantity calibration of the air which may be incompletely known. samples. Mycotoxin Respirator A class of fungal metabolites that have a toxic A personal protection device designed to effect on animals and humans (e.g., protect the wearer from inhalation of 42

55 hazardous substances in the atmosphere. Stachybotrys A genus of toxigenic moulds (Hyphomycetes) Sampling characterized by producing slimy heads of A process of taking an aliquot for warted, ellipsoidal, usually black conidia from representative analyses. clusters of inflated phialides (flask-shaped fertile cells). The toxins produced by SAS Stachybotrys chartarum (= atra) are extremely Surface Air System sampler. potent macrocyclic trichothecenes. Stachybotrys lives primarily on damp cellulose. SBS Sick building syndrome. According to World Health Organization's definition, consists of a Substrate Substance on or in which a fungus is living. group of non-specific symptoms such as eye, nose, or throat irritation; a sensation of dry mucous membranes; dry skin, rash; mental Thermophilic Organisms that prefer a temperature above fatigue; headache; nausea; dizziness; coughing; 37EC for growth and survival. Such agents can hoarseness; wheezing; or itching and non- grow in temperatures up to 58EC and are specific hypersensitivity reactions. isolated in hot springs, soil, compost piles, gas furnace humidifiers, etc. This may be an Settle plate A petri plate filled with microbial growth important group causing indoor air problems in buildings. medium and left open for a prescribed period of time so that bioaerosols can settle on it. It is then closed up and incubated to allow the Toxigenic A substance or biological entity that has the growth of colonies of fungi or bacteria. property itself or can produce one or more compounds that have the property to harm humans or other animals. Slit-to-agar sampler A vacuum/culture air sampling device. The Bourdillon slit-to-agar sampler draws air through a narrow slit onto the surface of a rotating petri dish filled with agar. As only one position on the agar surface is under the slit at one time, some idea of the temporal variation Trichothecene A class of toxins produced by certain fungal species such as Fusarium sporotrichoides and Stachybotrys chartarum (= atra). These mycotoxins cause severe health effects in in propagule intake can be obtained by humans and other animals (e.g., examining the plate. This in turn gives an idea deoxynivalenol, or DON; vomitoxin; nivalenol; of the degree of homogeneity of propagule T-2; HT-2; diacetoxyscirpenol, or DAS). distribution in the air itself. Vacuum/culture technique Air samplers that sample mould propagules by means of a vacuum drawing airborne matter onto culture media. VOC 43

56 Volatile organic compounds. Some VOCs are of industrial origin, such as compounds that Xerophilic Refers to fungi that prefer to grow in substrata evaporate from housekeeping or maintenance with low water activity. products used or in storage. Others are produced by certain micro-organisms, Yeast including 1-octen-3-ol, 2-pentanol, and Yeast is vernacular for unicellular fungal 3-methyl-2-butanol. organisms that reproduce mostly by budding, although some fission yeasts do exist. This Water activity term is of no taxonomic significance and is The molecular availability of water. Water useful only to describe a certain morphological becomes less available (lower in free energy) form of a fungus. Most true yeasts belong to when molecules are ordered by interactions one of the phyla of true fungi called with solute molecules or capillary surfaces. Ascomycota (some others in Basidiomycota and those not yet shown to have a sexual stage WHMIS in Deuteromycota). The characteristic sexual Workplace Hazardous Materials Information spores of Ascomycota occur in even numbers System. The legislation ensuring the worker's (normally eight) in a sac called an ascus. The right-to-know using three key elements: labels, asexual spore is a budding cell called a Material Safety Data Sheets, and worker blastoconidium. Yeasts may be pathogenic, training. such as Candida albicans, C. tropicalis, and Cryptococcus neoformans. Other yeasts are of industrial importance, such as baker's yeast for household or brewing use. 44

57 APPENDIX A Human Health Effects of Indoor Fungi 45

58 Human Health Effects of Indoor Fungi A.1 Introduction 1 5 Several excellent reviews have compiled the myriad illnesses caused by fungi. The list includes the major categories of cancer, infection, toxic diseases, and immunologic diseases. Mechanisms of pathogenesis are equally eclectic. Fungi produce potent mycotoxins, allergens, and biologically active cell wall components, and spores can induce polyclonal cell activation in vitro. 1 In most public and private buildings, indoor concentrations of fungi are lower than outdoor concentrations, and the species mix is similar. Thus, the health risks of fungal exposure should not be increased indoors. However, in some buildings there are environments that promote the growth of fungi, resulting in higher indoor concentrations and a different species mix. This situation has been associated with adverse health effects in several case reports, usually of extremely high exposure to toxigenic species. Apart from these isolated case reports, the population health significance of indoor fungal contamination is unknown. Mould amplifiers are frequently reported by occupants of homes in North America and Europe. In a large questionnaire study of Canadian homes, reported indoor dampness and/or mould growth were positively associated with reported asthma and respiratory and non-specific symptoms. If this association were causally related, indoor dampness and/or moulds would be responsible for a significant amount of respiratory illness in the general population. Improved building structure and function would effectively reduce dampness and improve the health of our society. Because of its potential significance to public health, research continues into clarifying the nature of the epidemiological association. This review summarizes the currently available information concerning the population health effects of indoor dampness and moulds and assesses the strength of evidence for causality. A.2 Methods Used to Search the Literature Data bases searched included Medline ( ), Embase ( ), and BIOS ( ). Keywords used were [(fungi or mould or mycotoxin) and (respiration or pulmonary or breath or inhalation) and (indoor air or public housing or public area or public building or workplace or work place)]. To verify completeness of the search, the keyword indoor air pollution was used by itself for Medline, but this did not contribute further relevant articles. Several articles and abstracts were obtained by personal communication with investigators active in the area of indoor mould and dampness. The following types of literature were included: journal articles, reviews, conference proceedings, technical reports, monographs, and meeting abstracts. The references of the reviews were also searched for further relevant articles. No studies of human health effects of indoor moulds and dampness were excluded. All are discussed with their strengths and weaknesses. Many poor quality studies may still provide useful pieces of information unaffected by the overall methodological weaknesses. The evidence used to assess causality was taken from the published series of McMaster University Clinical Epidemiology Rounds. 6 46

59 A.3 Fungi in Residential Buildings Epidemiological studies from several countries have demonstrated health effects associated with home dampness and moulds. The findings are remarkably consistent across different climates, societies, housing characteristics, and scientific investigators. To allow the reader to appreciate this consonancy, studies have been grouped by geographic region. A.3.1 United Kingdom Melia et al. administered questionnaires to mothers of 191 schoolchildren 5 6 years of age in northern England and measured relative humidity in the children's bedrooms and living rooms. 7 The prevalence of at least one respiratory symptom (cough, chest colds, wheeze, asthma, bronchitis) was 85% among boys and 64% among girls whose bedroom weekly mean relative humidity was at least 75%. Corresponding prevalences were approximately 58% and 45% where humidity was lower (p<0.05, boys only). Strachan et al. randomly sampled one in three Edinburgh primary schools Questionnaire information obtained for 1000 children 6 7 years of age demonstrated associations between interior bedroom wall dampness and wheeze, cough, and chest colds (p<0.01). The odds ratio between wheeze and bedroom mould was 3.00 after adjusting for housing tenure, household 8 smokers, crowding, and gas cooking. However, exercise-induced one-second forced expiratory volume (FEV1) decline, measured for 873 children, was not related to reported mould growth. 8 Measured bedroom humidity number (n) (n=778) and viable airspora (n=88) were not associated with respiratory symptoms or with exercise-induced FEV1 decline. Thus, the strong relations between questionnaire-reported respiratory symptoms and questionnaire-reported home dampness/mould could not be validated by objective measures of asthma, humidity, and mould growth. Either the questionnaire ascertainment of mould growth and symptoms created an artifactual relationship or the objective measures of asthma were less sensitive than the questionnaire: the within-subject coefficient of FEV1 variation was 8.5%, and only 40 of 873 children demonstrated an FEV1 decline of at least 20%. Relative humidity in a room may not accurately reflect damp (and mouldy) micro-environments. Airspora measurements are highly variable, as physical activity within the room will render settled dust and spores airborne. If species identification is not performed, the total spore count will represent a variable combination of toxigenic and non-toxigenic species. Using total spore counts with varying toxigenicity will obscure any existing association between toxigenic fungi and health. Martin and colleagues studied 358 tenants of Edinburgh flats constructed in the 1930s and s. Children's and adults' respiratory symptoms were obtained by questionnaire completed by an adult woman tenant. Inspectors measured the humidity inside the home and recorded signs of dampness, mould, and condensation. Damp homes were associated with more respiratory problems, aches and pains, diarrhea, and headaches among children but not among adults. At least one respiratory problem over the previous two months was reported in 85% of children living in damp homes as opposed to 60% in homes not considered damp. A subsequent and larger study (n=597 households) by the same investigators found associations for adults between home dampness and moulds and the following symptoms: bad nerves, 47

60 aching joints, nausea and vomiting, backache, blocked nose, fainting spells, constipation, and 13 breathlessness (p<0.05). The last symptom was also associated with indoor total air spore counts (p=0.019). No significant differences in psychological distress measured by questionnaire were found between residents living in damp and dry homes. 14 Hyndman studied 345 British Bengalis living in 60 publicly owned flats. Visible mould growth was present in one-half of the centrally heated homes and all of those non-centrally heated. Generally, respiratory and other non-specific symptoms, but not peak flows, were associated with average weekly humidity, temperature, and percent mould cover in the most severely affected room, with odds ratios approximating 2. Total spore counts, taken over 15 minutes, were associated only with reported depression. A.3.2 The Netherlands Waegemaekers and colleagues studied 328 adults and 190 children from 185 homes in a 15 Dutch coastal town. Mean measured spore concentrations were 192 colony-forming units per 3 cubic metre (CFU/m ) (geometric mean 2.8) in homes classified by questionnaire as damp and CFU/m (geometric mean 2.1) in homes classified as dry. When homes were classified as damp or dry, adjusted odds ratios for adults were greater than 1 for most respiratory symptoms and were statistically significant at p<0.01 for reported wheezing and allergy. No non-respiratory complaints were investigated, and the presence of allergy was not considered a potential confounder but simply an outcome variable. Brunekreef reported results from two studies of 6- to 12-year-old children carried out in (n=1051) and 1989 (n=3344). Damp stains were reported in 15 24% of homes, and mould growth was reported in 9 15%. Odds ratios ranged from 1.5 to 3.0 between cough and wheeze and dampness and mould growth after adjusting for parental education, household nitrogen dioxide level, and smoking. Maximum-mid expiratory flow (MMEF) was reduced 5.4% in homes with reported mould (p<0.1) compared with 1.0% in those without mould. Among the adult respondents of the 1991 survey, the odds ratio for the association between cough and either dampness or moulds approximated 2.0 (p<0.001), an effect size similar to that of current smoking. 17 Verhoeff provided evidence that the respiratory symptoms associated with home dampness may be mediated through allergic sensitization to dust mites and possibly moulds. 18 From a random sample of 7632 schoolchildren 6 12 years of age, 259 cases (defined by the presence of chronic cough or wheeze, or dyspnea and wheeze, or physician-diagnosed asthma) were compared with 257 controls (those without respiratory symptoms) on residential mould and dampness (as judged by both the tenant and an independent inspector). Odds ratios for dampness and mould approximated 1 2 for all cases and were approximately 0.5 higher for cases with elevated immunoglobulin type E (IgE) against dust mites and common moulds. However, between cough and reported mould, odds ratios increased to 6.4 (95% CI ) when restricted to those with elevated IgE to mites and/or moulds. To validate their methods, the investigators correlated the presence of respiratory symptoms with diminished FEV1, and the kappa statistics for agreement between reported and measured mould and/or dampness were

61 A.3.4 Sweden Holmberg studied 33 adults in 13 Swedish residences selected by mould and moisture 3 problems. Increased levels of airborne Aspergillus (>50 CFU/m ) were associated with increased 19 reports of eye and skin irritation, cough, phlegm, and common colds. A larger questionnaire study of 4990 Swedish children whose parents were non-smokers found dampness to be 20 associated with cough, as indicated by an odds ratio of 1.9 (p<0.05). Rylander et al. reported an 21 association between $-1,3-glucan and cough and pruritus. Five glucan measures were available, one from each of five buildings. Thus, the association may be confounded by other differences between the buildings or their occupants. A.3.5 United States 22 Brunekreef et al. studied 4625 children from six U.S. cities. Home dampness was indicated by a positive response to at least one of the following questions: (1) Does water ever collect on the basement floor? (2) Has there ever been water damage to the building? and (3) Has there ever been mould or mildew on any surface inside the home? The odds ratios between dampness and respiratory symptoms ranged from 1.23 (95% CI ) for persistent wheeze to 2.16 (95% CI ) for persistent cough after adjusting for maternal smoking, age, sex, city of residence, and parental education. The adjusted odds ratios between mould (question 3) and respiratory symptoms ranged from 1.40 (95% CI ) for non-chest illness to 2.12 (95% CI ) for cough. There was no statistically significant (p<0.05) association between spirometric measures and the dampness indicators. A.3.6 Canada In 1988, Health and Welfare Canada conducted a questionnaire-based study of Canadian communities. In total, parents or guardians of schoolchildren received a questionnaire, and (83%) questionnaires were returned. The reported prevalence of home dampness or moulds in Canadian homes was approximately 38%. The prevalence of lower respiratory symptoms (any of cough, phlegm, wheeze, wheeze with dyspnea) was increased among those who reported dampness/moulds. Among the children between five and eight years of age, the prevalences of lower respiratory symptoms were 19.5% and 13.2% in homes with and without reported dampness/moulds, respectively. The corresponding adjusted odds ratio was 1.50 (95% CI ). Among the adult non-smoking questionnaire respondents, lower respiratory symptoms were reported in 19% and 11% of those with and without reported exposure to dampness/mould. The odds ratio for all adults adjusted for smoking was 1.62 (95% CI ). 49

62 A.4 Fungi in Office Buildings Compared with residential settings, there are very few studies of exposure to fungi in office buildings. Although several buildings may contain thousands of employees, the number of buildings available for analysis is small. If no buildings with fungal contamination were included, health effects from fungi would not be expected. Skov and colleagues carried out a comprehensive examination of 14 municipal buildings in 26 Denmark. Questionnaire-reported symptoms were related to several factors, such as floor dust, floor coverings, crowding, building age, and ventilation, but not airborne micro-fungi. Generally, measurements were taken from one site in each of the buildings and generalized to the entire building and its occupants. The duration of sampling and growth media were not stated. Levels of 3 airborne fungi were unremarkable, between 0 and 111 CFU/m. 27 Harrison and colleagues reported a study of 15 office buildings in Great Britain. Using a six-stage Andersen sampler, the following median fungal counts were obtained: approximately CFU/m in the four naturally ventilated buildings, and about 30 CFU/m in the remainder. Although symptom prevalences were lower in naturally ventilated buildings, a positive association was reported between symptom prevalences and counts of fungi and bacteria. Tamblyn et al. studied four mechanically ventilated high-rise office buildings in Montreal 28 during the spring or fall of Within each building, the ventilation was randomized six times to either 20 or 50 cubic feet per minute per person for one week. Increased ventilation sometimes increased and sometimes decreased the spore counts. The resulting changes in viable spore counts explained 36 64% of the variation in symptoms (p=0.06 for the Pearson and p=0.20 for the Spearman correlation coefficient). This association was seen in only one building, and varying the ventilation rate also influences many other air quality characteristics, making any change difficult to attribute to any one cause. In this population, a second study design was employed, comparing employees reporting work-related symptoms with the remainder of the employees. Fungal spore 3 counts were 24 CFU/m higher in the latter than the former group. Recently, 43 of 49 New York employees exposed to satratoxin H.-producing Stachybotrys atra in an office building (> viable spores per cubic centimetre in sheetrock walls) 29 underwent a health examination. Complaints involved the general areas of respiratory, fatigue, central nervous system, and skin and eye irritation. IgE specific for Stachybotrys atra was detected in four subjects. No control subjects were mentioned, making it impossible to determine if there was an exposure health association in this descriptive study. 50

63 A.5 Summary of the Literature There were 23 publications in the English-language literature from the United 7 14,26, , ,28 Kingdom, the Netherlands, Sweden, the United States, Canada, and 27 Denmark. These represented 17 unique populations; in several cases there was more than one 9 10,16 17, ,16,18,20 22,24,25 paper per population. Twelve studies included children, and 11 included 12 15,17,19,23,26 29 adults. The majority of studies identified an association between self-reported respiratory symptoms and exposure, whether assessed by the resident, building inspector, or spore count. In contrast, objective measures of respiratory illness have not been associated with exposure. A.5.1 Exposure Assessment In the majority of studies, exposure took place in private dwellings. Much less published 21,26 28 information was available from public buildings. Eight studies assessed exposure to home 8,9,15,16,18,20,22,23 dampness and moulds by questioning the home resident. Questions used included signs of dampness or mould, damp spots, evidence of water damage, a history of flooding, stale odour, and the presence of a wet crawl space, silver-fish, or sow-bugs. Six studies used 12 14,16,18,20 independent inspectors, who assessed homes for the characteristics just listed. Three 7,10,14 11,13 15,19,21,26 28 studies measured relative humidity, and nine studies measured airspora. In 3 Edinburgh, approximately 50 species or genera were isolated, with 50% of total CFU/m being Penicillium and Cladosporium. Total spore counts ranged from 0 to , with the median 3 11 being about 200 CFU/m. Another study identified the common fungi as Penicillium, 3 15 Aspergillus, Botrytis, and Cladosporium, with counts ranging between 34 and 2000 CFU/m. One other study identified Aspergillus, Cladosporium, and Penicillium, also with median total 3 19 spore counts of approximately 230 CFU/m. A.5.2 Health Outcomes In all studies, self-reported respiratory symptoms were ascertained. These generally included cough, wheeze, asthma, bronchitis, and colds going to the chest. Occasionally, hay fever and sore throat were included. Four studies attempted objective confirmation of respiratory 14 16,22 9 illness, either by resting spirometry or peak flows or by exercise-induced FEV reduction ,19,22,23,28 Eight studies assessed self-reported non-respiratory symptoms, which included aches and pains, vomiting, diarrhea, bad nerves, headache, and fever. A.5.3 Results Among children, all studies detected associations between self-reported symptoms and self-reported mould/dampness. Among adults, similar associations were found in six of seven 13 15,17,19, residential studies and one of three office studies. Sample sizes ranged between and , with at least five studies having more than 1000 subjects. When exposure was selfreported, associations were always found with respiratory symptoms. The same was 51

64 true for the presence of other symptoms. In contrast to self-reported symptoms, objective indicators of respiratory illness have never been shown to be associated with self-reported mould and dampness. Although no one respiratory symptom was outstanding in its strength of association with self-reported exposure, cough and wheeze were consistently related and often had the strongest odds ratios, usually ranging between 1.5 and 3. In one study, the odds ratios increased in cases with increased serum-specific IgE to the house dust mite Dermatophagoides 18 pteronyssinus and the moulds Penicillium, Alternaria, Aspergillus, or Cladosporium. Similar to self-reported exposures, studies assessing exposure by a building inspector showed associations 12,13,14,16,18 with respiratory symptoms. Two of three studies using relative humidity demonstrated 7,14 13,15,19,21 associations with respiratory symptoms. Four of five residential studies and one of three office studies measuring airspora demonstrated associations with respiratory symptoms. A.6 Arguments Supporting a Causal Association Between Indoor Air Fungi and Population Health A.6.1 Fungi Cause Disease Mediators of disease include mycotoxins, allergens, biologically active cell wall components, and polyclonal cell activators. Antigenic properties of fungi have been implicated in asthma, allergic bronchopulmonary aspergillosis, extrinsic allergic alveolitis, and humidifier fever. Stachybotrys atra, a hydrophilic mould that can produce highly toxic macrocyclic trichothecenes, was thought to be responsible for chronic health problems in a family dwelling. The disease symptoms were consistent with a toxin etiology, toxins were isolated from the air, and workers became ill while removing contaminated materials. Other adverse human health effects from mycotoxin inhalation, documented in uncontrolled case reports, include renal failure, tremorgenic encephalopathy, organic dust toxic syndrome, and non-specific complaints including headache, sore throat, alopecia, flu symptoms, diarrhea, fatigue, dermatitis, 12,13,34 malaise, cough, rhinitis, epistaxis, and fever. Fungal volatiles, typically short-chained alcohols and aldehydes, account for mouldy odour: 1-octen-3-ol has a mushroom odour, 2-octen- 1 1-ol smells musty, and geosmin has an earthy smell. Reaction to exposure is variable, from none 38 to illness, but data on their health effects are scarce. $-1,3-glucan, a constituent of fungal cell 21 walls, may be related to dry cough and irritation of the skin, eye, and throat. Relevant to the residential setting, Tarlo et al. reported that 14 of 26 allergic subjects (rhinitis, asthma) tested positive, in skin prick tests, to fungi in their homes. 32 A.6.2 Toxigenic Fungi Are Present in Indoor Air Mycotoxin-producing fungi are not uncommon in residential buildings. Smith et al. employed the MRC-5 monolayer cultures to assay mycotoxins in 83 fungal isolates from damp 39 public housing in Edinburgh. Forty-seven percent of isolates were considered toxigenic, causing 12 51% cell mortality. In a study of 52 Canadian homes, evidence of mycotoxin production was found in three homes containing Aspergillus fumigatus

65 Trichothecene mycotoxins have also been isolated from the ventilation system of three sick buildings in Montreal. 41 Epidemiological findings are consistent across geographic regions. Similar health effects have been consistently found in both children and adults and by different investigators in different countries using different questionnaires. This association has usually been found whether exposure to dampness and moulds is ascertained by questioning a resident, building inspection, or 23,24 enumerating spores. The Canadian study also showed a dose response effect. The odds ratio for cough was 1.61 (95% CI ) for the presence of one versus no mould sites and 2.26 (95% CI ) for two versus no mould sites. A.7 Arguments Against a Causal Association Between Indoor Air Fungi and Population Health Study designs have been cross-sectional in nature, with no evidence for temporality or reversibility. Home dampness and moulds have usually been assessed by the occupant's response to questions concerning flooding, water damage, and visible moulds in the house. Although dampness promotes mould growth, it may also indicate inadequate home ventilation and increased levels of several indoor air contaminants, from allergens to combustion products. Dampness also promotes dust mites, which can cause respiratory disease. Bacterial endotoxin has been implicated in several cases of building-related illness (BRI) involving humidifier reservoirs contaminated with 31 gram-negative bacteria. The majority of studies have ascertained symptoms by questionnaire, which poses doubts. First, the existence of a group of respondents who generally over-report and another group of respondents who generally under-report could result in an artifactual relationship if those who report more respiratory symptoms also report more home dampness and moulds, and vice versa. Second, subjects ill for other reasons may be more likely than those who are symptom-free to report the presence of home dampness and moulds in an attempt to explain their health problems. This could also result in an artifactual relationship. However, Brunekreef, contrasting symptom reporting with objective measures of lung function, concluded that bias or 16 confounding was unlikely. The Canadian study, however, did not find evidence for (but cannot exclude) this bias; whether or not subjects had been diagnosed as having allergies or asthma, the observed association between symptoms and dampness was not influenced. Finally, the measured relative risks were usually small, less than 2, and therefore relatively susceptible to confounding. A.8 Summary and Conclusion Fungi can and do cause a myriad of diseases. Potentially pathogenic fungi are not uncommon in the indoor environment, and diseases caused by indoor fungi have been documented in case reports. The burden of illness in the population attributable to fungi in private homes and public buildings is not yet known. Epidemiological studies have consistently detected an association between respiratory symptoms and home dampness and mould growth, but causality in these studies has not been established. Until the magnitude of the population risk is known, it would be prudent, based on current evidence, to remediate indoor sources conducive to fungal growth. 53

66 A.9 References 1. Flannigan, B., McCabe, E.M., and McGarry, F. Allergic and toxigenic micro-organisms in houses. J. Appl. Bacteriol., 70 (Suppl.): 618 (1991). 2. Burge, H. Bioaerosols: prevalence and health effects in the indoor environment. J. Allergy Clin. Immunol., 86: 687 (1990). 3. Tobin, R.S., Baranowski, E., Gilman, A., Kuiper-Goodman, T., Miller, J.D., and Giddings, M. Significance of fungi in indoor air: report from a working group. Can. J. Public Health, 8 (Suppl.): 51 (1987). 4. Jarvis, B.B. Mycotoxins and indoor air quality. In: Biological contaminants in indoor environments. P.R. Morey, J.C. Feeley, Jr., and J.A. Otten (eds.). ASTM Spec. Tech. Publ. 1071, American Society for Testing and Materials Committee D-22 on Sampling and Analysis of Atmospheres, Section 06 on Biological Aerosols (1990). 5. Broder, I. Primer on airborne fungi and other microorganisms for safety officers of Human Resources Development Labour Component. Final report to Technical Services Division, Occupational Safety and Health Branch, Labour Component, Human Resources Development, Government of Canada, 29 March (1994). Contract: 1993, File YR Department of Clinical Epidemiology and Biostatistics, McMaster University Health Sciences Centre. Clinical Epidemiology Rounds. How to read clinical journals: 4. To determine etiology or causation. Can. Med. Assoc. J., 124: 985 (1981). 7. Melia, R.J.W., Florey, C. du V., Morris, R.W., Goldstein, B.D., John, H.H., Clark, D., Craighead, I.B., and Mackinlay, J.C. Childhood respiratory illness and the home environment. Association between respiratory illness and nitrogen dioxide, temperature and relative humidity. Int. J. Epidemiol., 11: 164 (1982). 8. Strachan, D.P. and Elton, R.A. Relation between respiratory morbidity in children and the home environment. Fam. Pract., 3: 137 (1986). 9. Strachan, D.P. Damp housing and childhood asthma: validation of reporting symptoms. Br. Med. J., 297: 1223 (1988). 10. Strachan, D.P. and Sanders, C.H. Damp housing and childhood asthma; respiratory effects of indoor air temperature and relative humidity. J. Epidemiol. Commun. Health, 43: 7 (1989). 54

67 11. Strachan, D.P., Flannigan, B., McCabe, E.M., and McGarry, F. Quantification of airborne moulds in the homes of children with and without wheeze. Thorax, 45: 382 (1990). 12. Martin, C.J., Platt, S.D., and Hunt, S.M. Housing conditions and ill health. Br. Med. J., 294: 1125 (1987). 13. Platt, S.D., Martin, C.J., Hunt, S.M., and Lewis, C.W. Damp housing, mould growth, and symptomatic health state. Br. Med. J., 298: 1673 (1989). 14. Hyndman, S.J. Housing dampness and health amongst British Bengalis in East London. Soc. Sci. Med., 30: 131 (1990). 15. Waegemaekers, M., Van Wageningen, N., Brunekreef, B., and Boleij, J. Respiratory symptoms in damp homes. Allergy, 44: 1 (1989). 16. Brunekreef, B. Associations between questionnaire reports of home dampness and childhood respiratory symptoms. Sci. Total Environ., 127: 79 (1992). 17. Brunekreef, B. Damp housing and adult respiratory symptoms. Allergy, 47: 498 (1992). 18. Verhoeff, A.P. Home dampness, fungi and house dust mites, and respiratory symptoms in children. Doctoral thesis (ISBN /CIP), University of Rotterdam, Rotterdam, The Netherlands (1994). 19. Holmberg, K. Indoor mould exposure and health effects. In: Indoor air '87, Proceedings of the 4th International Conference on Indoor Air Quality and Climate. B. Seifert, H. Esdorn, M. Fischer, H. Rden, and J. Wegner (eds.). Institute for Water, Soil, and Air Hygiene, Berlin. p. 637 (1987). 20. Andrae, S., Axelson, O., Björkstén, B.,FrPderiksson, M., and Kjellman, N-I.M. (eds). Symptoms of bronchial hyperreactivity and asthma in relation to environmental factors. Arch. Dis. Child., 63: 473 (1988). 21. Rylander, R., Persson, K., Goto, H., Yuasa, K., and Tanaka, S. Airborne beta-1,3-glucan may be related to symptoms in sick buildings. Indoor Environ., 1: 263 (1992). 22. Brunekreef, B., Dockery, D.W., Speizer, F.E., Ware, J.H., Spengler, J.D., and Ferris, B.G. Home dampness and respiratory morbidity in children. Am. Rev. Respir. Dis., 140: 1363 (1989). 23. Dales, R., Burnett, R., and Zwanenburg, H. Adverse health effects in adults exposed to home dampness and molds. Am. Rev. Respir. Dis., 143: 505 (1991). 55

68 24. Dales, R.E., Zwanenburg, H., Burnett, R., and Franklin, C.A. Respiratory health effects of home dampness and molds among Canadian children. Am. J. Epidemiol., 134: 196 (1991). 25. Dekker, C., Dales, R.E., Bartlett, S., Brunekreef, B., and Zwanenburg, H. Childhood asthma and the indoor environment. Chest, 100: 922 (1991). 26. Skov, P., Valbjo/rn, O., Pedersen, B.V., and the Danish Indoor Climate Study Group. Influence of indoor climate on the sick building syndrome in an office environment. Scand. J. Work Environ. Health, 16: 363 (1990). 27. Harrison, J., Pickering, C.A.C., Faragher, E.B., and Austwick, P.K.C. An investigation of the relationship between microbial and particulate indoor air pollution and the sick building syndrome. Respir. Med., 86: 225 (1992). 28. Tamblyn, R.M., Menzies, R.I., Comtois, P., Hanley, J., Tamblyn, R.T., Farant, J.P., and Marcotte, P. A comparison of two methods of evaluating the relationship between fungal spores and respiratory symptoms among office workers in mechanically ventilated buildings. In: Healthy buildings, Proceedings of the ASHRAE IAQ '91 Conference, Washington, DC. p. 136 (1991). 29. Johanning, E., Jarvis, B.B., and Morey, P.R. Clinical-epidemiological investigation of health effects caused by Stachybotrys atra building contamination. In: Indoor air '93, Proceedings of the 6th International Conference on Indoor Air Quality and Climate. Vol. 1. Health effects. J.J.K. Jaakkola, R. Ilmarinen, and O. Seppänen (eds.). Helsinki. p. 225 (1993). 30. Salvaggio, J. and Aukrust, L. Mold-induced asthma. J. Allergy Clin. Immunol., 68: 327 (1981). 31. Hodges, G.R., Fink, J.N., and Schlueter, D.P. Hypersensitivity pneumonitis caused by a contaminated cool-mist vaporizer. Ann. Intern. Med., 80: 501 (1974). 32. Tarlo, S.M., Fradkin, A., and Tobin, R.S. Skin testing with extracts of fungal species derived from the homes of allergy clinic patients in Toronto, Canada. Clin. Allergy, 18: 45 (1988). 33. Sorenson, W.G., Frazer, D.G., Jarvis, B.B., Simpson, J., and Robinson, V.A. Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra. Appl. Environ. Microbiol., 53: 1370 (1988). 34. Croft, W.A., Jarvis, B.B., and Yatawara, C.S. Airborne outbreak of trichothecene toxicosis. Atmos. Environ., 20: 549 (1986). 56

69 35. Di Paolo, N., Guarnieri, A., Loi, F., Sacchi, G., Mangiarotti, A.M., and Di Paolo, M. Acute renal failure from inhalation of mycotoxins. Nephron, 64: 621 (1993). 36. Gordon, K.E., Masotti, R.E., and Waddell, W.R. Tremorgenic encephalopathy: a role of mycotoxins in the production of CNS disease in humans. Can. J. Neurol. Sci., 20: 237 (1993). 37. Yoshida, K., Ando, M., and Araki, S. Acute pulmonary edema in a storehouse of moldy oranges: a severe case of the organic dust toxic syndrome. Arch. Environ. Health, 44: 382 (1989). 38. Samson, R.A. Occurrence of molds in modern living and working environments. Eur. J. Epidemiol., 1: 54 (1985). 39. Smith, J.E., Anderson, J.G., Lewis, C.W., and Murad, Y.M. Cytotoxic fungal spores in the indoor atmosphere of the damp domestic environment. FEMS Microbiol. Lett., 100: 337 (1992). 40. Miller, J.D., Laflamme, A.M., Sobol, Y., Lafontaine, P., and Greenhalgh, R. Fungi and fungal products in some Canadian houses. Int. Biodeterior. Bull., 24: 103 (1988). 41. Smoragiewicz, W., Cossette, B., Boutard, A., and Krystyniak, K. Trichothecene mycotoxins in the dust of ventilation systems in office buildings. Int. Arch. Occup. Environ. Health, 65: 113 (1993). 57

70 APPENDIX B Indoor Air Quality in Office Buildings: A Technical Guide, Health Canada, 1993 (Pages 48 49)

71 Indoor Air Quality in Office Buildings: A Technical Guide, Health Canada, 1993 (Pages 48 49) Interpretation of Results. Since 1989, the ACGIH Bioaerosols Committee has recommended rank order assessment as a means of interpreting air sampling data. This interpretation has been part of the practice in Government of Canada investigations since The presence of one or more species of fungi indoors, but not outdoors, suggests the presence of an amplifier in the building. Species identification is critical to the analysis. Because of the problems noted above, numerical guidelines cannot be used as the primary determinant of whether there is a problem. However, numerical data are useful under defined circumstances. Information from a large data set obtained by experienced individuals using the same instrument has practical value. Investigations of more than 50 federal government buildings over several years has resulted in the creation of such a data set. Fungal data from about 600 samples taken between 1986 and 1991 with a Reuter centrifugal sampler with a 4-minute sampling time have been used to prepare the interpretation notes shown below. Data acquired with other samplers require similar analysis. However, if a 4-minute sampling time is used, the numerical data from any proprietary sampler will probably be comparable. Significant numbers of certain pathogenic fungi should not be present in indoor air (e.g., Aspergillus fumigatus, Histoplasma, and Cryptococcus). Bird or bat droppings in air intakes, ducts or rooms should be assumed to contain these pathogens. Action should be taken accordingly. Some of these species cannot be measured by air sampling techniques. The persistent presence of significant numbers of toxigenic fungi (e.g., Stachybotrys atra, toxigenic Aspergillus, Penicillium, and Fusarium species) indicates that further investigation and action should be taken accordingly. The confirmed presence of one or more fungal species occurring as a significant percentage of a sample in indoor air samples and not similarly present in concurrent outdoor samples is evidence of a fungal amplifier. The normal air mycoflora is qualitatively similar and quantitatively lower than that of outdoor air. In federal government buildings, the 3-year average has been approximately 3 40 CFU/m for Cladosporium, Alternaria, and non-sporulating basidiomycetes. More than 50 CFU/m may be reason for concern if there is only one species other than 3 Cladosporium or Alternaria present. Further investigation is necessary. 59

72 3 Up to 150 CFU/m is acceptable if there is a mixture of species reflective of the outdoor air spores. Higher counts suggest dirty air filters or other problems. 3 Up to 500 CFU/m is acceptable in summer if the species present are primarily Cladosporium or other tree and leaf fungi. Values higher than this may indicate failure of the filters or contamination in the building. The significant presence of fungi in humidifiers and diffuser ducts and on mouldy ceiling tiles and other surfaces requires investigation and remedial action regardless of the airborne spore load. There are certain kinds of fungal contamination not readily detectable by the methods discussed in this report. If unexplained SBS [sick building syndrome] symptoms persist, consideration should be given to collecting dust samples with a vacuum cleaner and having them analysed for fungal species. 60

73 APPENDIX C Standard Operating Procedure: An Example for the Investigation and Remediation of Indoor Air Quality Contamination by Mycological Agents

74 Standard Operating Procedure: An Example for the Investigation and Remediation of Indoor Air Quality Contamination by Mycological Agents Any procedures involving direct handling and manipulating of potentially contaminated materials should be assigned only to trained personnel as required by WHMIS [Workplace Hazardous Materials Information System] and the provincial Occupational Health and Safety Acts. These individuals must have received information on the possible hazards as well as effective strategies to protect building occupants and themselves. They must also be reminded to wear appropriate personal protective equipment prior to entering contaminated areas. In small scale operations, such as 0.3 square metres or less, gloves and masks with good fit and proper seal may be used. The affected material should be decontaminated prior to removal. For intermediate scale operations such as 3 square metres or less, gloves and half-face respirators are advised. It should be noted that half-face respirators and gloves are strongly recommended for remediation at any scale if the presence of toxigenic fungi is known or suspected. Procedures that involve large scale operations, such as 10 square metres, will result in the disturbance of contaminated materials. Redistribution of these contaminants by the HVAC (heating, ventilation, and air conditioning) system could also follow. Under such circumstances, the area must be physically isolated (especially with respect to ventilation) by sheets of plastic and taping. Engineering controls such as a negative pressure air unit equipped with a HEPA (high-efficiency particulate air) filter and vacuum cleaners with HEPA filters are required especially when remediation is on a large scale. Evacuation of building occupants during such procedures must be considered. A full disposable suit with head to toe coverage, including a full face respirator equipped with HEPA cartridges, is acceptable. It is important to note that a chlorine removal cartridge is required with the use of high concentrations of bleach. When dealing with highly toxic materials, extra precautions must be exercised during removal of the personal protective equipment to avoid exposure. All equipment and other surfaces (such as the floor, ceiling, or the plastic drapes) within the segregated areas must, prior to removal, be surface decontaminated by treating with a 10% solution of household bleach (laundry or household bleach contains approximately 5.25% available chlorine before dilution) with an optional addition of % non-ionic detergent for an enhancement of cleansing and penetrating actions. Contaminated materials should be double bagged and tied for disposal by incineration. A thorough cleaning and decontamination of the areas immediately adjacent are also recommended. Sampling attachments and tools must be decontaminated between each assay in order to avoid cross contamination of samples, rooms and/or buildings. If there is a necessity, during the investigative stage, to transport a sample of the suspected contaminant to another location for diagnostic or consultational purposes, it must first be rendered innocuous. Alternatively, when one is not certain that the sample is 62

75 dangerous, it should be packaged and transported as a diagnostic specimen under the TDG [Transportation of Dangerous Goods] Act and Regulations. However, if the sample has been identified as dangerous to human health and safety, then it must be packaged and transported by a TDG certified individual under the classification of 6.2. Reference New York City Department of Health, New York City Human Resources Administration, and Mount Sinai Irving J. Selikoff Occupational Health Clinical Center. Guidelines on assessment and remediation of Stachybotrys atra in indoor environments. New York, NY (1993). 63

76 APPENDIX D Sampling Methodology for Fungal Bioaerosols and Amplifiers in Cases of Suspected Indoor Mould Proliferation

77 Sampling Methodology for Fungal Bioaerosols and Amplifiers in Cases of Suspected Indoor Mould Proliferation Problems attributed to moulds in indoor air are usually associated with on-site proliferation of the moulds in the affected building. Only occasionally are mould conidia from an outdoor source suspected of being potential sources of symptoms. Moulds proliferating within a building usually do so in discrete locations where moisture and substrate conditions are conducive. Typical sites of indoor mould proliferation are damp cellulosic materials (e.g., wallboard, wallpaper, carpet backing, damp papers); debris in ventilation ducts, in carpets, or in mattresses or upholstered furniture; poorly maintained humidifiers; insulation on which organic film has accumulated; constantly humid painted, caulked, or plastic surfaces (e.g., windowsills, shower stalls, cold air return vents); and potted plant soils. The ultimate goals of diagnostic indoor air mould studies are: to determine if sufficient mould propagules, particularly those bearing irritating or immunosensitizing chemical components, are being produced and dispersed within the building to account for (or predict) symptomatology; and if a connection between moulds and symptoms is likely, to find and eliminate sites of mould amplification within the building. These goals are usually addressed through one or both of the following strategies: air sampling (or sometimes dust sampling) to determine the level and types of viable fungal propagules within building trouble spots, and to suggest the presence of significant indoor mould growth sites ( amplifiers ); and physical search of likely problem areas to detect conspicuous mould amplifiers. Further details of these two strategies are outlined below. D.1 Sampling for Fungal Bioaerosols D.1.1 Major Types of Mycological Air Sampling Techniques Air sampling for fungal structures is, at the most fundamental level, divided into techniques based on the culture of live propagules and techniques based on the trapping and visualization of living or dead materials. This appendix deals primarily with the former type of sampling methodology. This is because many situations of suspected indoor air contamination involve toxigenic, allergenic fungal genera with small, nondescript conidia, such as Penicillium and Aspergillus. These are often difficult to assess accurately with particle-trapping devices such as Rotorod samplers and spore traps, where culturing cannot be done and analysis of samples tends to be biased towards the identification of larger, distinctively shaped, and/or dark-pigmented structures. Common fungi possessing such large 65

78 or dark propagules, such as Cladosporium, Alternaria, Pithomyces, and Bipolaris, are often relatively innocuous fungi, predominantly coming from outdoor sources. Other conspicuous allergens, such as basidiospores of the bracket fungus Ganoderma applanatum and ustilaginaceous smut teliospores, may also be counted with unparalleled accuracy, but these are once again from outdoor sources and have little relevance to the major questions assessed by the Working Group. It should be noted, however, that Kozak et al. found a Rotorod sampler to be very useful for visualizing non-viable Stachybotrys, Ulocladium, and Alternaria conidia 1 emanating from contaminated carpets in homes. Although these authors actually located and identified the problem moulds by a combination of inquiries about water damage, site inspection, and direct sampling from suspect surfaces, they felt that the Rotorod could be an important component of a detailed evaluation. They recommended its use in combination with an Andersen sampler for viable propagules, plus rigorous site examination, history taking, and direct sampling. The air sampling techniques elucidating viable propagules can be grouped into two categories: those relying on gravity to effect sedimentation of the mould propagules onto growth medium, and those based on pumping a measured amount of air onto or through a propagule collecting device. The sedimentation plate is archetypical of the former category, whereas the latter category contains a variety of sampling devices. The former will be dealt with first. D.1.2 Settle (Sedimentation) Plates A large number of publications substantiate the fact that settle plates elucidate a biased 2 sample of the viable airborne mould propagules. The reasons for this are twofold. First, propagules have differential settling rates according to their weight and aerodynamic form. Settle plates are particularly efficient at detecting large conidia in indoor air, whereas the proportion of conidia belonging to important small-spored genera such as Aspergillus and Penicillium is underestimated. Second, whereas pumping samplers cause some air disturbance, settle plates are still. Some disturbance is usually necessary in air sampling to resuspend settled conidia, which would ordinarily become airborne under conditions of normal human activity in the rooms being 3 investigated. Actual normal human activity substantially improves fungal isolation, even where pump samplers are used. Despite these limitations, settle plates are still widely used, at least in preliminary studies, in remote or impoverished areas, or as an adjunct to physical searching for amplifiers. As semiquantitative samplers, when adequately exposed (a commonly used protocol is for one hour at 4 tabletop level under conditions of ordinary room activity), they can readily be used to discern the likely presence of significant indoor mould amplifiers (except in special cases, e.g., Stachybotrys amplifiers). A problematic indoor mould amplifier, if it consists of small-spored fungi such as Aspergillus or Penicillium, produces a sufficiently large quantity of airborne propagules that these species show up as a significant proportion of the isolates occurring on a settle plate. The gravitational bias against these smaller conidia is compensated for by the high numbers of conidia produced by any significant amplifier (subject to normal disturbance). 66

79 Some specific sampling deficiencies have been attributed to settle plates by researchers investigating parameters not relevant to the detection of significant indoor amplifiers. The strength of this technique lies in the fact that epidemiologically important toxigenic or allergenic fungi, unlike invasively pathogenic fungi, must be present in large quantities. The quantitative species distribution of fungi growing in any habitat tends to have an inverse exponential distribution, in which there are very large numbers of individuals representing a few predominant taxa and very small numbers of individuals representing each of an indefinitely large number of minor taxa. 5,6 2 Hence, to point out that settle plates tend to grow fewer taxa than impacted air plates or that 7 they detect members of a particular fungal group in fewer sites than do impacted air samplers is not necessarily of practical significance. With adequate exposure times, especially under conditions of typically low air turbulence indoors, only members of the asymptotic tail of minor taxa are strongly likely to be missed in any given habitat. The best general definition of an adequate exposure time is that necessary to obtain an adequate representation of the smallest spore type of practical interest. In indoor studies, this spore type is often the Aspergillus/Penicillium conidium; the present author finds that the majority of one-hour, indoor settle plates he receives from putatively mould-affected buildings have members of these taxa as predominant species. This ineluctable but anecdotal finding needs to be followed up by more definitive comparative studies. Published comparative studies between volumetric and gravitational techniques often have inadequacies. Sayer et al. compared 15-minute samples taken by means of a vacuum sampler drawing 28.3 L of air per minute with gravity plates exposed for an entirely inadequate, and only 2 desultorily parallel, 15 minutes. Solomon, in a better-designed study, found that 30-minute settle plate samples showed very little correlation with 1- to 10-minute Andersen volumetric samples, and that numerically predominant, small-spored taxa were sometimes missed or very poorly 8 represented. Verhoeff et al., however, found a strong correlation between Andersen samples and 4,9 duplicate 60-minute settle plate samples in two different studies. The earlier of these two 9 studies showed that the number of species isolated on settle plates on conventional high water activity medium (malt extract agar) was not significantly different from that obtained with four 4 major types of volumetric air samplers. The later study showed that settle plates isolated significantly fewer species than the Andersen sampler but did not comment on whether these species were relatively abundant or uncommon. No study to date has deviated from the prevailing focus on abundance and commented on the extent to which different types of samples allowed the recognition of synecological patterns signifying the presence and types of indoor fungal amplifiers. Such patterns (e.g., Penicillium brevicompactum and Aspergillus versicolor usually signifying moist but not currently saturated wall covering paper) can be seen even in a relatively light deposition of smaller-spored types on a settle plate (or in a light outgrowth of low-viability spore types in either gravitational or volumetric sampling) and are sufficient to direct further on-site investigation. Either a heavy or a light deposition of such a pattern indicates the likely presence of a larger or smaller, closer or more distant, exposed or more concealed, but in any case undesirable mould proliferation site. Notwithstanding the serviceability of longer settle plate exposures in detecting these patterns, however, settle plates are best used in combination with a thorough initial site inspection to detect any macroscopically visible 67

80 mould growth. (Because of low-viability propagule types like Stachybotrys, the same caution holds for volumetric samples.) Volumetric air sampling should be regarded as superior and used whenever it can be made available. In Canada, the species predominant in indoor mould amplifiers ordinarily form a small or insignificant proportion of spora in outdoor air samples (except near large compost sites such as municipal leaf dumps or where abundant dust from stored crops or wood is encountered). Common examples of fungi strongly associated with indoor proliferation are Aspergillus versicolor, A. fumigatus, A. niger, members of Penicillium subgenus Penicillium (with a few exceptions), and black-spored Scopulariopsis species. Receipt of settle plates predominantly colonized by significant numbers of such fungi is an excellent indicator of potentially problematic indoor mould amplification. Accompanying outdoor air control plates, exposed sufficiently far away from the building studied to avoid outflow of building bioaerosols, are strongly recommended: they are characteristically negative for these fungi. False negative or ambiguous settle plates may be obtained from buildings with very restricted mould amplifiers, with very still air in undisturbed rooms, with amplifiers of poorly culturable species (e.g., Stachybotrys chartarum [= atra]), or with amplifiers consisting of species with poor airborne dissemination (e.g., Aureobasidium on windowsills, Cladosporium on painted cold air vents, Fusarium and many other wet-spored fungi from indoor plants, and possibly Chaetomium). The health effects of the species with low aerial dispersal have been suggested to 10 be insignificant, but, as intermittent or cumulative airborne dispersal of desiccated material may occur, some wet-spored species may be quite significant. Stachybotrys is an example of a wetspored fungus for which significant airborne dissemination and health effects are well substantiated. Also, noxious volatiles may be produced by some wet-spored fungi. Little is known, however, about the health effects of the volatiles of wet-spored indoor fungi; many such species are not odoriferous to ordinary olfaction. With settle plates, as with any other culturing of airborne moulds, the most informative level of interpretation usually requires that the analyst be able to distinguish among the predominant species and species-groups of the genus Penicillium, Aspergillus, and other relatively complex fungal groups. Also, if outdoor air controls are inadequate, the analyst must be sufficiently familiar with the local ecology of moulds and yeasts to detect deviations from their ordinary seasonal frequency in outdoor air. In Toronto, for example, a high number of Penicillium subgenus Aspergilloides colonies on a household settle plate in winter is an excellent indicator of mould proliferation indoors; the same finding in September might be non-diagnostic. Such interpretation of uncontrolled samples requires a mycologist with some aerobiological baseline data. As meaningful analysis of settle plates is based primarily on recognizing the synecological assemblage of isolates consistent with the presence of indoor mould amplifiers and is only secondarily concerned with the actual numbers of colonies detected, the problem of establishing acceptable and unacceptable numbers of colonies in indoor samples cannot be addressed. Any actions taken against indoor mould proliferation must therefore be triggered by factors other than the demonstration of a threshold count. Locating and examining any mould amplifiers not detected in preliminary inspection are logical follow-up steps once settle plates have revealed that these amplifiers are present. The substrate nature of the amplifier 68

81 can usually be read from the settle plate. For example, Stachybotrys indicates very moist cellulose, often in previously flooded or soaked material, not uncommon in sheltered areas behind wallpaper or wallboard paper growing in contact with the glue. Penicillium chrysogenum suggests crumbs; Eurotium suggests, among other things, carpets with accumulations of dry skin scales and dust; Aspergillus versicolor suggests humid wallboard or other humid cellulose, including cellulosic dust within ducts; and so on. In practice, common indications for characterization and remediation of the discovered amplifiers are occurrence of symptoms consistent with adverse reaction to indoor moulds and/or building management or administrative concerns that such amplifiers might cause symptoms in future, might indicate or exacerbate degradation of materials, or might cause offence owing to noxious odours or to the cosmetic, aesthetic, psychological, or political disadvantages of harbouring conspicuous decay. Once established mould amplifiers have been demonstrated, managers usually find themselves under strong pressure to clean them up. D.1.3 Vacuum/Culture (Pump) Samplers Pump samplers for viable propagules can be broken down into (1) samplers impacting a stream of air onto a fungal medium surface; (2) samplers trapping propagules from an airstream in a viscous fluid, which can then be plated on growth medium; and (3) samplers trapping propagules on a membrane filter, which can be eluted onto growth medium. In category #1 are slit samplers such as the New Brunswick slit-to-agar sampler, sieve impactors such as the Andersen and SAS samplers, and centrifugal impactors such as the RCS (details of sampling with these devices are outlined by Muilenberg; also, see Glossary for information about Andersen, RCS, and slit-to-agar samplers; see Verhoeff et al. for a chart showing the air intake rates, usual sampling times, and particle size biases of volumetric and non-volumetric sampling devices and 9,11 techniques). In category #2 are liquid impingers and modifications of slit samplers to deposit 12 propagules in easily melted glycerol/gelatin gels. In category #3 are various assemblages of pumps and filter cassettes drawing measured quantities of air through membrane filters impervious to fungal conidia. A considerable literature exists comparing the efficacy of these samplers. Indeed, until recently, such studies have greatly predominated over other kinds of studies that might have predictive value in the analysis of indoor mould problems for example, studies of the biological effects of exposure to mould conidia, or studies of the composition of fungal communities associated with indoor proliferation and related symptoms. Even though much useful information has been gathered by the analysis of sampling devices and accurate sampling is important, an overemphasis on this essentially non-biological topic is deleterious. At present, even if propagule concentrations in room atmospheres were known with absolute accuracy, we would be little further ahead in understanding the association (if any) between these numbers and symptoms, and we would not be assisted in the location or remediation of mould proliferation sites. In any case, correlative statistics should allow any moderately imperfect but reasonably consistent sampler to yield numbers that could be meaningfully gauged against symptoms, toxin levels, and a variety of related topics. These numbers must be broken down by fungal group, not given as total colonyforming units (CFU), as the 69

82 totality of spora includes varying proportions of potentially irritating and relatively benign particles. (Imagine, as a comparison, trying to gauge the chance of acquiring malaria simply by counting total mosquitoes in any habitat.) Several recent studies have been performed comparing the sampling efficiencies of different pump samplers. Most of these studies have embodied some uncontrolled variables: for example, some fail to standardize the sampling durations and volumes, and many generate data by sampling in unpredictably non-homogeneous room air. They must therefore be interpreted with considerable caution, and those interested in this topic are advised to do a more detailed review than can be accomplished here. A few recent studies are worthy of mention, but the conclusions mentioned below should not necessarily be taken at face value. Buttner and Stetzenbach analysed the efficiencies of Andersen six-stage, SAS, and Burkard (suction slit impactor for direct examination of particles) samplers and settle plates in a controlled room with known 3 concentrations of Penicillium chrysogenum conidia. The Andersen sampler gave the most accurate and consistent results, but differences between it and the other volumetric samplers were marginal. Verhoeff et al. did comparative field trials in houses with the slit-to-agar, single-stage 9 (N6) Andersen, RCS, and SAS samplers. The slit-to-agar sampler and the Andersen were 13 concluded to be the most precise. This study, however, was criticized because it did not take into account the variation attributable to air mass discontinuity over the different sampling times used. (Total volume of air sampled was standardized at the expense of varying sampling duration.) Smid et al. similarly compared the single-stage Andersen, slit, RCS, and SAS 14 samplers. Once again, the Andersen and slit samplers were reported to give the best results, with RCS reasonably comparable; SAS underestimated CFU counts by about 50%. These authors concluded that the RCS remained useful because of its convenience of use and acceptable accuracy. In heavily contaminated environments (e.g., barns), Andersen samplers may suffer from overexposure, with multiple propagules being counted as one after impaction via the same sieve hole and with subsequent colony overgrowth. Correction factors have been published for moderately overexposed plates but are inadequate for heavily overexposed plates. Diminishing the sampling time is a possible response but has the disadvantage that spatial/temporal discontinuities in airborne mould propagule distribution may skew results. For example, a 30-second exposure may fortuitously sample a current of relatively clean air from a window draft not generally representative of a contaminated room; or, likewise, a short exposure may sample the peak of a burst of conidia from a disturbed mould amplifier or reservoir and may show numbers well above those typically found in the room. For this reason, devices trapping propagules in liquid may be more accurate in heavily contaminated environments. With such samplers, sampling times can be longer without overwhelming the analytical capabilities of the system. Thorne et al. found that both impinger samples and eluted Nucleopore filters were more accurate than Andersen samples 15 in barns housing swine. Blomquist et al. modified a slit sampler to deposit spores on agar or glycerol/gelatin gels and then homogenized or liquefied these gels and plated them out in a classic 12 dilution series. When glycerol/gelatin gels were used, results were comparable to those obtained using eluted Nucleopore filters. 70

83 Impingers have not been widely accepted in ordinary indoor mould sampling work. Most potentially problematic airborne moulds have highly water-repellent conidia that, in contact with aqueous media, tend to adhere to surface films and hydrophobic surfaces and to clump together in 11 minute air pockets. Trapping of such hydrophobic particles in impingers is not efficient. A comparative study of Andersen and impinger samplers from a hospital under renovation showed that impingers underestimated CFU by 90% or more (R.C. Summerbell, Ontario Ministry of Health, pers. commun.). In conclusion, for public buildings, various slit, sieve, and centrifugal samplers should give comparable results. Absolute propagule count is not a realistic sole criterion for building remediation, as large counts from outdoor air are possible at some times of year, particularly in buildings with openable windows or with air filters that do not exclude smaller fungal conidia. The most efficient use of samplers, arguably, should be to detect conidial shedding by indoor mould amplifiers. Although such shedding may very well result in high CFU counts, and although high counts in general will be more significant than low counts, certain factors may cause a truly problematic amplifier to yield low to moderate counts for example, sampling at a distance from the amplifier, misleading air distribution patterns, and low conidial viability. Pasanen et al. found that viable spore counts were sometimes less than 25% of the total spores detected by scanning electron microscopy in farm and urban homes. Other difficulties are outlined by Miller. Such information strongly argues on behalf of using air sampling as a detector of amplifiers and a semiquantitative indicator of approximate bioaerosol density rather than as an absolute arbiter of indoor air standards. The relevance of spora counts is greatest where there is a diffuse and difficult-to-access amplifier present in a building. Two recurrent examples are mould growth in ventilation ducts in buildings with self-contained air recirculation systems and moulds apparently associated with a multiplicity of lightly and sporadically contaminated books in a library. In these cases, the idea of finding a discrete amplifier and eliminating it, the practical solution for the great majority of indoor mould problems, may be problematic. Although heavily contaminated ducts or books must clearly be cleaned up or otherwise dealt with (as must ducts or books with confirmed Stachybotrys colonization), the possibility of lightly contaminated environments is evident. The traditional question of determining an air contamination level requiring action is relevant in these instances. Clearly, it simplifies matters to restrict the analysis only to those fungi associated with the amplifiers and to exclude fungi known to be associated with any incident outdoor air. (It is unlikely that indoor and outdoor types will have an additive effect, as their toxin chemistry and antigenicity will be largely distinct; the possibility of additive glucan effects needs further investigation.) The difficulty is to know what factor to correlate numbers of indoor-generated mould propagules with in order to assign health significance to the findings. Essential dose response information needed to correlate numbers of fungal propagules of particular chemical composition to health effects in humans is absent for all moulds, and, as tolerance to moulds appears to vary biologically among individuals and appears to relate at least partially to the vagaries of allergic sensitization, acceptable dose information would doubtless be arduous to acquire even if ethical tests could be devised. Surrogate tests such as in vitro tests for the responses of human cells (e.g., alveolar macrophages) are in their infancy, and animals lack the ability to corroborate or disconfirm the persistent, subjective 71

84 symptoms commonly reported in cases of indoor mould proliferation. The need for objective measures of adverse responses to mould inhalation is great, and devising such measures would be an important step in coming up with scientific correlates between spore counts and the need for remediation of buildings. This appendix will not discuss direct air or dust sampling methods for fungal biochemicals. Methods detecting general fungal materials such as chitin, glucan, and ergosterol lack the ability to discriminate between fungal elements from indoor and outdoor sources. Hence, they will tend to give unambiguously interpretable single-case results (as opposed to multi-case statistical trends) only in cases where there is an extreme indoor build-up or where indoor accumulation of outdoor fungal material is otherwise known to be insignificant. Tests detecting specific toxins or volatiles may be very useful, but in their specificity they are beyond the scope of this document. See Miller for a summary of some limitations of sampling for volatiles. 17 Dust, carpet, or surface swab samples may serve in place of air samples but may contain many normally settled elements (e.g., Mucorales; also Fusarium other than predominant species on local agricultural crops), which in many cases are not significantly present in the air. On the other hand, dust samples have the advantage of containing a relatively long-term record of the history of fungal deposition within a building and may thus relieve investigators of problems posed by the bioaerosol variability of different air currents seen in short-duration air samples. Further investigation is needed to give criteria for the interpretation of dust sample results, but results to date show some promise. Swabs may play a useful role in the sampling of patches of mould growth that have been detected visually, but they are inferior to surface scrapings, as they tend to select spores and leave conidiophores/pycnidia/ascomata behind. D.1.4 Media Used in Sampling The media used in sampling fungal air and dust spora are diverse. They fall into several distinct categories: generally permissive media of high water activity, designed to allow growth and in situ identification of a wide range of fungi (e.g., Sabouraud, 2% malt extract agar, V-8 agar); generally permissive media with components restricting colony diameter ( restrictive media ), minimizing colony overgrowth and allowing in situ identification of at least some fungi (e.g., rose bengal agar, various high water activity media containing dichloran, Littman oxgall agar); media of low water activity, with or without factors restrictive of colony diameter, for isolation of moderately osmotolerant to xerophilic fungi (e.g., dichloran 18% glycerol agar, Czapek's + 40% sucrose agar, 2% malt extract agar + 10% salt [ malt and salt agar]); and media selective for particular groups of fungi ( selective media, e.g., Sabouraud/cycloheximide medium for the majority of human pathogens, Onygenales, Herpotrichiellaceae, and Ophiostomatales; media with benomyl for Basidiomycetes, Zygomycetes, Endomycetes, Pleospora/Cochliobolus anamorphs, and Microascaceae). At least two apparently irreconcilable dichotomies must be addressed by the person trying to select a single medium for an indoor fungal study: first, that no one medium will optimize growth of both the significant fungi adapted to high substrate water activity (e.g., Stachybotrys) and the significant fungi requiring lowered water activity 72

85 (e.g., Eurotium, Wallemia); and second, that the best media for identifying organisms in situ are also the most problematical for colony overgrowth and formation of spurious satellite colonies in shipping and handling. Currently, the two most widely used media for general sampling are malt extract agar (MEA) and dichloran 18% glycerol agar (DG18). The former was recommended by the American 18 Conference of Governmental Industrial Hygienists (Burge et al.), while the latter has been 9 shown to be useful in a variety of recent studies (e.g., Verhoeff et al.). The limitations of MEA are that it allows extensive colony overgrowth and supports osmophilic fungi poorly; DG18 supports osmophiles well and facilitates growth of the moderately osmotolerant fungi that form the majority of indoor species of concern (Penicillium, Aspergillus), but it causes poor growth in 19 moderately osmointolerant fungi such as Scopulariopsis (van Reenen-Hoekstra et al.) and may support Stachybotrys and other highly osmointolerant species poorly or not at all (R.A. Samson, pers. commun.). A near-ideal sampling protocol might include both media. Malt and salt agar may be a good alternative for DG18: long used for isolation of osmotolerant fungi, it also allows growth of Stachybotrys chartarum (J.D. Miller, pers. commun.). The colonies of this fungus are restricted but are readily seen. In general, workers who, for practical reasons, prefer to use a single medium must be mindful of the types of fungi they will be excluding from their data. Because most indoor environments contain a variety of osmophilic Aspergillus species, DG18 often tends to isolate the greatest number of species in comparison trials (e.g., Verhoeff et 9 al.) ; if a single medium must be chosen, DG18 may be optimal, but it should not be used alone except in combination with thorough visual and microscopic visual search to detect excluded fungal types, especially Stachybotrys. Such physical searching is recommended for Stachybotrys in any case, since Stachybotrys may be predominantly represented in the environment by non-viable conidia. Despite this, Stachybotrys is not uncommonly obtained in air samples, and any investigator wishing to maximize the likelihood of detecting significant Stachybotrys amplifiers is obliged to consider the use of air sampling with an appropriate medium. The present author uses Littman oxgall agar extensively, primarily because of its tendency to repress sporulation and prevent satellite colony formation and colony overgrowth during shipping and handling in transit from test sites to the laboratory. It grows Stachybotrys well, has been observed to grow Eurotium (Aspergillus glaucus and allies) in high numbers in at least some cases (R.C. Summerbell, unpublished data), grows Wallemia occasionally (but probably not in a good representation of its true population predominance), and does not grow Aspergillus restrictus and allies. In its only formal comparison test as an indoor mould sampling medium, it showed significantly fewer colonies than three other media, including Sabouraud agar, at the sixth 20 day of incubation (Morring et al.). The authors conceded, however, that this time period was too short for a full evaluation. Littman showed that the eponymous medium outperformed 21 Sabouraud agar over longer incubation periods. Littman oxgall agar, however convenient it may be for shipping, requires much labour, since the majority of colony types must be subcultured for identification. Further, they must be subcultured soon after plates are received since, as nonsporulating colonies, they may become non-viable within 2 3 weeks. This medium would therefore be a 73

86 suboptimal choice for anyone doing his/her own sampling and mycology or receiving plates or strips within a day or two of exposure. Rose bengal agar or its dichloran-supplemented variant are also restrictive of overgrowth and, while delaying or repressing sporulation to a lesser extent than Littman oxgall agar, may be relatively robust in shipping while permitting a relatively high level of in situ identification. It must be borne in mind that rose bengal generates high-energy oxygen species on exposure to light, and illuminated medium may become lethal to fungi. Dichloran rose bengal agar has grown 9 significantly fewer colonies than other media in at least one study, although this effect was not 14 observed in others (e.g., Smid et al.). Unfortunately, Verhoeff et al. did not record the time 9 period allowed for incubation. Restrictive media in general slow colony growth rates, and, for a fair biological (as opposed to purely practical) trial, such media should be observed only after sufficient incubation to ensure maximal colony outgrowth. An in-house trial showed that Littman oxgall agar gave colony numbers equivalent to those obtained with MEA in hospital renovation air samples incubated for 14 days (R.C. Summerbell, unpublished data). The fact that all existing fungal sampling media have recognized shortcomings is a further blow against the former aerobiological ideal of using a perfected, standardized sampling device with a perfected, standardized growth medium to evaluate potential fungal aerosol problems with reference to standard guidelines for acceptable numbers of CFU. This ideal, which was always predicated on the reduction of all members of the three major terrestrial fungal phyla to a single, alchemical mass substance, clearly must yield to the reality of biological diversity. As more becomes known about the actual hazardous substances associated with airborne fungal materials, methods indicating the occurrence or likely occurrence of these substances will be developed. In the meantime, the investigator engaged in detecting potentially significant amplifiers must simply ensure that an adequate diversity of techniques is used to cover the diversity of possible amplifiers. D.2 Direct Detection of Amplifiers Procedures for the direct detection of mould amplifiers may be used either after an air sample has predicted the presence of amplifiers or as a preliminary survey. Common places where significant amplifiers can be visually identified are in water-damaged walls on or under wallpaper or wallboard paper (whether painted over or not), on the backings of water-damaged carpets, on heating, ventilating, and air conditioning (HVAC) coils, pans, vanes, and so on, on damp papers (e.g., after flood, including floods created by fire-fighting operations), within walk-in refrigerators and incubators, and in any moist organic materials, including any moist object composed of cellulose. Amplifiers may also be visible on windowsills, as well as shower stalls and washroom 7 fixtures. These windowsill and washroom amplifiers, if small and not involving cellulosic material (i.e., moulds growing only on paint, ceramic, grouting, or plastic), are seldom problematic. More extended amplifiers in these situations may be problematic, and even hypersensitivity pneumonitis, which normally requires long-term heavy exposure to develop, has on rare occasion been linked to heavy growth of fungi in shower or other washroom amplifiers. 74

87 Additional amplifiers may be detected by microscopic sampling. Common practices are the transparent tape sampling of duct interiors, slide examination of humidifier basin materials, and examination of small fibre samples cut or pulled away from filters or carpet backings in which mould growth is suspected. Further direct detection of amplifiers may be performed by culturing for example, plating out of humidifier fluids and plating out of scrapings or swabs from recognized or suspected mould growth on walls or other surfaces. D.3 References 1. Kozak, P.P., Gallup, J., Commins, L.H., and Gillman, S.A. Currently available methods for home mold surveys. II. Examples of problem homes surveyed. Ann. Allergy, 45: 167 (1980). 2. Sayer, W., Shean, D.B., and Ghosseiri, J. Estimation of airborne fungal flora by the Andersen sampler versus the gravity settling culture plate. J. Allergy, 44: 214 (1969). 3. Buttner, M.P. and Stetzenbach, L.D. Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling. Appl. Environ. Microbiol., 59: 219 (1993) [erratum notice: Appl. Environ. Microbiol., 59: 1694 (1993)]. 4. Verhoeff, A.P., van Wijnen, J.H., Brunekreef, B., Fischer, P., van Reenen-Hoekstra, E.S., and Samson, R.A. Presence of viable mold propagules in indoor air in relation to house damp and outdoor air. Allergy, 47: 83 (1992). 5. Good, I.J. The population frequencies of species and the estimation of population parameters. Biometrika, 40: 237 (1953). 6. Gochenaur, S.E. Fungi of a Long Island oak birch forest. II. Population dynamics and hydrolase patterns of the soil penicillia. Mycologia, 76: 218 (1984). 7. Hyvärinen, A., Reponen, T., Husman, T., Ruuskanen, J., and Nevalainen, A. Composition of fungal flora in mold problem houses determined with four different methods. In: Indoor air '93, Proceedings of the 6th International Conference on Indoor Air Quality and Climate. Vol. 4. Particles, microbes, radon. P. Kalliokoski, M. Jantunen, and O. Seppänen (eds.). Helsinki. p. 273 (1993). 8. Solomon, W.R. Assessing fungus prevalence in domestic interiors. J. Allergy Clin. Immunol., 56: 235 (1975). 9. Verhoeff, A.P., van Wijnen, J.H., Boleij, J.S.M., Brunekreef, B., van Reenen-Hoekstra, E.S., and Samson, R.A. Enumeration and identification of airborne viable mold propagules in houses: a field comparison of selected techniques. Allergy, 45: 275 (1990). 75

88 10. Kapyla, M. Frame fungi on insulated windows. Allergy, 40: 558 (1985). 11. Muilenberg, M.L. Aeroallergen assessment by microscopy and culture. Immunol. Allergy Clin. N. Am., 9: 245 (1989). 12. Blomquist, G., Palmgren, V., and Ström, G. Improved techniques for sampling airborne fungal particles in highly contaminated environments. Scand. J. Work Environ. Health, 10: 253 (1984). 13. Miller, J.D. Fungi and the building engineer. In: IAQ '92, Environments for people. M. Geshwiler (ed.). ASHRAE, Atlanta, GA (1993). 14. Smid, T., Schokkin, E., Boleij, J.S., and Heederik, D. Enumeration of viable fungi in occupational environments: a comparison of samplers and media. Am. Ind. Hyg. Assoc. J., 50: 235 (1989). 15. Thorne, P.S., Kiekenhaefer, M.S., Whitten, P., and Donham, K.J. Comparison of bioaerosol sampling methods in barns housing swine. Appl. Environ. Microbiol., 58: 2543 (1992). 16. Pasanen, A.L., Kalliokoski, O., Pasanen, P., Salmi, T., and Tossavainen, A. Fungi carried from farmers' work into farm homes. Am. Ind. Hyg. Assoc. J., 50: 631 (1989). 17. Miller, J.D. Fungi as contaminants in indoor air. Atmos. Environ., 26A: 2163 (1992). 18. Burge, H.A., Chatigny, M., Feeley, J., Kreiss, K., Morey, P., Otten, J., and Petersen, K. Guidelines for assessment and sampling of saprophytic bioaerosols in the indoor environment. Appl. Ind. Hyg., 9: 10 (1987). 19. van Reenen-Hoekstra, E.S., Samson, R.A., Verhoeff, A.P., van Wijnen, J.H., and Brunekreef, B. Detection and identification of moulds in Dutch houses and non-industrial working environments. Grana, 30: 418 (1991). 20. Morring, K.L., Sorenson, W.G., and Attfield, M.D. Sampling for airborne fungi: a statistical comparison of media. Am. Ind. Hyg. Assoc. J., 44: 662 (1983). 21. Littman, M.L. Growth of pathogenic fungi on a new culture medium. Tech. Bull. Reg. Med. Tech., 18: 409 (1948). 76